KR20230125236A - Nucleic Acids Encoding Anchor Modified Antibodies and Uses Thereof - Google Patents

Abstract

An anchor-modified immunoglobulin polypeptide is described herein wherein the anchor directs the immunoglobulin polypeptide to a receptor of interest. Anchor-modified immunoglobulin polypeptides are generally characterized as having an anchor at the N-terminus, eg, the receptor binding portion of a ligand that binds the receptor. Non-human animals that have been genetically modified with recombinant immunoglobulin segments encoding anchor-modified immunoglobulin polypeptides are capable of making anchor-modified immunoglobulin polypeptides. Such non-human animals are also provided along with methods and compositions for making and using non-human animals. Methods of producing anchor-modified immunoglobulins from non-human animals are provided, as well as anchor-modified immunoglobulins produced therefrom.

Description

Nucleic Acids Encoding Anchor Modified Antibodies and Uses Thereof

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63/129,893, filed on December 23, 2020, and U.S. Provisional Application No. 63/219,402, filed on July 8, 2021 and each of these applications is incorporated herein by reference.

sequence listing

The sequence listing recorded in file 10507WO01_ST25.txt is 47 kilobytes, was created on December 17, 2021, and is incorporated herein by reference.

Monoclonal antibody products have revolutionized the biopharmaceutical industry and have made significant advances in the treatment of many diseases. Despite these advances and the knowledge gained from the use of monoclonal antibodies for therapeutic applications, diseases persist that involve monoclonal antibodies binding and/or difficult-to-access targets, highlighting the need for a variety of approaches to develop effective therapeutics. do.

Disclosed herein is an improved in vivo method for identifying and developing new antibody-based therapeutics, in some embodiments, antibodies (eg, monoclonal antibodies and/or fragments thereof) that can be used for the treatment of a variety of diseases. It is recognized that it is desirable to manipulate non-human animals as a system. Nucleic acids, non-human animals, methods and polypeptides disclosed herein relate to anchor-modified immunoglobulins. Anchors as described herein generally include a receptor binding portion of a non-immunoglobulin polypeptide that binds a cognate receptor. An anchor appended to an immunoglobulin helps to increase the affinity of the immunoglobulin for the anchor's cognate receptor, thereby improving the immunoglobulin's binding properties. Nucleic acid molecules that encode anchor-modified immunoglobulins and/or can be used to modify non-human animals such that the non-human animals can make de novo anchor-modified immunoglobulins are described herein.

The anchor-modified immunoglobulins described herein are at least partially by variable region (V) segments, e.g., immunoglobulin (Ig) heavy chain variable region (V _H ) segments or Ig light chain variable region (V _L ) segments. can be encoded and modified to encode anchors between and at operative linkages of the Ig leader sequence and the framework (FR) and complementarity determining region (CDR) sequences of the germline V segment.

Accordingly, targeting vectors and nucleic acid molecules comprising non-human animal genomes, comprising unrearranged or rearranged modified Ig V segments, are also described. Also described are non-human animal genomes, non-human animal cells, and non-human animals comprising the nucleic acid molecules described herein.

A recombinant nucleic acid molecule described herein may comprise a modified immunoglobulin (Ig) variable (V) segment encoding an anchor modified Ig polypeptide, wherein the modified Ig V segment comprises a nucleic acid sequence encoding an Ig signal peptide and a nucleic acid sequence encoding an anchor between framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and a nucleic acid sequence encoding CDR3 of a germline Ig V segment or variant thereof; wherein the anchor-modified Ig polypeptide comprises (i) an Ig signal peptide, (ii) an anchor, and (iii) FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment or variant thereof in operative linkage. wherein the anchor comprises a receptor binding portion of a non-immunoglobulin polypeptide of interest that binds a cognate receptor, optionally wherein the recombinant nucleic acid molecule lacks any other V segments. Recombinant nucleic acid molecules described herein may also include one or more rearranged (non-rearranged) Ig diversity (D) segments, one or more rearranged (non-rearranged) Ig binding (J) segments, and/or one or more Ig constant regions (C ) gene.

In some embodiments, the Ig signal peptide is an Ig signal peptide from a germline Ig V segment, or variant thereof. In some embodiments, the Ig signal peptide comprises the sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7). In some embodiments, the anchor comprises a linker connecting the receptor binding portion of the non-immunoglobulin polypeptide of interest to FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment or variant thereof. In some embodiments, the linker comprises the sequence sequence GGGGS (SEQ ID NO: 5).

In some embodiments, the germline Ig V segment or variant thereof is a germline Ig heavy chain variable (V _H ) segment or variant thereof, e.g., a human (h) germline Ig V segment or variant thereof, e.g., Germline Human (h) Segment V _H 1-2, Segment Germline hV _H 1-3, Segment Germline hV _H 1-8, Segment Germline hV _H 1 18, Segment Germline hV _H 1-24, Germline segment hV _H 1-45, germline segment hV _H 1-46, germline segment hV _H 1-58, germline segment hV _H 1-69, germline segment hV _H 2-5, germline segment hV _H 2-26 segment, germline hV _H 2-70 segment, germline hV _H 3-7 segment, germline hV _H 3-9 segment, germline hV _H 3-11 segment, germline hV _H 3 segment 13, germline hV _H segment 3-15, germline hV _H segment 3-16, germline hV _H segment 3-20, germline hV _H segment 3-21, germline hV _H segment 3-23, germline hV _H segment 3-30; germline hV _H 3-30-3 segment, germline hV _H 3-30-5 segment, germline hV _H 3-33 segment, germline hV _H 3-35 segment, germline hV _H 3-38 segment, reproduction germline hV _H 3-43 segment, germline hV _H 3-48 segment, germline hV _H 3-49 segment, germline hV _H 3-53 segment, germline hV _H 3-64 segment, germline hV _H 3- 66 segments, germline hV _H 3-72 segments, germline hV _H 3-73 segments, germline hV _H 3-74 segments, germline hV _H 4-4 segments, germline hV _H 4-28 segments, germline hV _H 4-30-1 segment, germline hV _H 4 30-2 segment, germline hV _H 4-30-4 segment, germline hV _H 4-31 segment, germline hV _H 4-34 segment, germline hV _H 4-39 segments, germline hV _H 4-59 segments, germline hV _H 4-61 segments, germline hV _H 5-51 segments, germline hV _H 6-1 segments, germline hV _H 7-4 -1 segment, germline hV _H 7-81 segment, or a variant thereof. In some embodiments, the germline Ig V segment or variant thereof is a germline hV _H 1-69 segment or variant thereof. In some embodiments, a variant is an allelic variant.

In some embodiments, a recombinant nucleic acid molecule may comprise a heavy chain variable region locus, e.g., 5' to 3' in operative linkages: (I) a modified Ig V _H segment; (II) one or multiple Ig heavy chain diversity (D _H ) segments, and (III) one or multiple Ig heavy chain binding (J _H ) segments. In some embodiments, the one or plurality of Ig D _H segments of (II) comprises one, plurality, or all human Ig D _H segments, and/or the one or plurality of Ig J _H segments of (III) comprises one , multiple, or all human Ig J _H segments. In some embodiments, the one or plurality of Ig D _H gene segments of (II) and the one or plurality of Ig J _H gene segments of (III), wherein the recombinant nucleic acid molecule is a modified Ig V _H gene segment and a rearranged Ig D gene segment. Recombined to include the _H /J _H sequence 5' to 3' in an operative linkage to form a rearranged Ig D _H /J _H sequence.

In some embodiments, modified Ig V _H gene segments and rearranged Ig D _H /J _H sequences are recombined to yield rearranged Ig V _H /D _H /J _H sequences encoding anchor modified Ig heavy chain variable domains. wherein the anchor modified Ig heavy chain variable domain comprises (i) an Ig signal peptide encoded by the rearranged Ig V _H /D _H /J _H sequence, (ii) an anchor, and (iii) FR1, a complementarity determining region (CDR) 1, FR2, CDR2, FR3, CDR3, and FR4 in operative linkages.

In some embodiments, the modified Ig V _H segment is an unrearranged modified Ig V _H gene segment.

In some embodiments, a recombinant nucleic acid disclosed herein further comprises a nucleic acid sequence encoding an Ig heavy chain constant region (CH ₎ , wherein the nucleic acid sequence encoding an Ig _CH is (I) a modified Ig V _H segment; (II) one or multiple Ig D _H segments, and (III) operably linked downstream of one or multiple Ig J _H segments. In some embodiments, the nucleic acid sequence encoding Ig _CH is an Igμ gene encoding an IgM isotype, an Igδ gene encoding an IgD isotype, an Igγ gene encoding an IgG isotype, an Igα gene encoding an IgA isotype, and/or an Igε gene encoding an IgE isoform. In some embodiments, a recombinant nucleic acid molecule described herein comprises a nucleic acid sequence encoding an anchor-modified Ig heavy chain, wherein the anchor-modified Ig heavy chain comprises (i) an Ig signal peptide, (ii) an anchor, (iii) ) an Ig heavy chain variable domain comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _H /D _H /J _H sequence, and (iv) an Ig C _H operable include in connection In some embodiments, the Ig _CH is a non-human Ig _CH , eg, a rodent Ig _CH , eg, a rat Ig _CH or a mouse Ig _CH .

In some embodiments, the germline Ig V segment or variant thereof is a germline Ig light chain variable (V _L ) segment or variant thereof. In some embodiments, a recombinant nucleic acid molecule may comprise a light chain variable region locus, e.g., in an operative linkage and 5' to 3': (I) a modified Ig V _L segment , and (II) one or multiple Ig light chain linkage (J _L ) segments.

In some embodiments, the modified Ig V _L segment and one or plurality of Ig J _L segments are recombined to form a rearranged Ig V _L /J _L sequence encoding an anchor modified Ig light chain variable domain, wherein the anchor modification The modified Ig light chain variable domain comprises FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by (i) an Ig signal peptide, (ii) an anchor, and (iii) a rearranged Ig V _L /J _L sequence. includes as an operable link.

In some embodiments, a recombinant nucleic acid molecule may comprise a nucleic acid sequence encoding a light chain variable region locus and an Ig light chain constant region ( _CL ), wherein the nucleic acid sequence encoding the Ig _CL is downstream and operable (I) modified Ig V _L segments and (II) one or multiple Ig light chain linking (J _L ) segments. In some embodiments, an anchor-modified Ig light chain comprises (i) an Ig signal peptide, (ii) an anchor, (iii) FR1, CDR1, FR2, CDR2, FR3 encoded by a rearranged Ig V _L /J _L sequence. , an Ig light chain variable domain comprising CDR3, and FR4, and (iv) an Ig C _L in operable linkage. In some embodiments, the Ig _CL is a non-human Ig _CL , eg, a rodent Ig _CL , eg, a rat Ig _CL or a mouse Ig _CL .

In some light chain variable region locus embodiments, the germline Ig V _L segment or variant thereof is a germline Ig light chain variable kappa (Vκ) segment or variant thereof. Accordingly, in some embodiments, a recombinant nucleic acid molecule described herein comprises, 5' to 3' in operative linkage: (I) a modified Ig Vκ segment, and (II) one or multiple Ig light chain linkages. Kappa (Jκ) segment. In some embodiments, a recombinant nucleic acid molecule described herein comprises, 5' to 3' in operative linkage: (I) a modified Ig Vκ segment, (II) one or multiple Ig light chain linked kappa (Jκ) ) and (III) a nucleic acid sequence encoding an Ig light chain constant kappa region (Cκ).

In some light chain variable region locus embodiments, the germline Ig V _L segment or variant thereof is a germline Ig light chain variable lambda (Vλ) segment or variant thereof. Thus, in some embodiments, a recombinant nucleic acid molecule described herein comprises, in operative linkage, 5' to 3': (I) a modified Ig Vλ segment and (II) one or a plurality of Ig light chain binding lambdas. (Jλ) segment. In some embodiments, a recombinant nucleic acid molecule described herein comprises, in operative linkage, 5' to 3': (I) a modified Ig Vλ segment, (II) one or multiple Ig light chain binding lambdas (Jλ). ) segment, and a nucleic acid sequence encoding the Ig light chain constant lambda region (Cλ).

In some embodiments, a recombinant nucleic acid molecule described herein comprises a sequence set forth as SEQ ID NO: 8 or a degenerate variant thereof, or SEQ ID NO: 10 or a degenerate variant thereof.

Non-human animals, including targeting vectors, non-human animal cells (eg, host cells, embryonic stem cells, etc.), and nucleic acid molecules are also described.

Targeting vectors comprising the recombinant nucleic acid molecule examples disclosed herein are also described. In some targeting vector embodiments, the targeting vector is such that, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus converts the non-human Ig C _H at the non-human Ig heavy chain locus. optionally further comprising 5' and 3' homology arms targeting the non-human Ig heavy chain locus for inclusion in an operative link upstream, wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and /or the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, a deletion of endogenous Ig V _H , D _H , and/or J _H gene segments, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule substitutes a non-human V _H segment at the non-human Ig heavy chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule comprises one or more non-human V _H segments, all non-human D H segments, and all non-human D _H segments at the non-human Ig heavy chain locus. Substitute all non-human J _H segments. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule _is one non-human V H segment, all non-human V _H segments, all non-human Ig heavy chain loci at the non-human Ig heavy chain locus. Substitute all but non-human D _H segments, and all non-human J _H segments. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus recombines operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus. Contains nucleic acid molecules. In some embodiments, a targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and a non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus is non-human Ig heavy chain locus. -contains 5' and 3' homology arms targeting a non-human Ig heavy chain locus to include a recombinant nucleic acid molecule operably linked to a human Ig heavy chain regulatory sequence, optionally wherein the non-human Ig heavy chain locus is a rodent or Endogenous rodent Ig heavy chain loci in rodent cells (eg, rodent embryonic stem cells) and/or non-human Ig heavy chain loci are human or humanized immunoglobulin heavy chain variable regions, endogenous Ig V _H , D _H , and/or J _H gene segment deletion, or a combination thereof, optionally wherein upon homologous recombination between non-human Ig heavy chain loci, the recombinant nucleic acid molecule comprises at least one non-human V _H segment at the non-human Ig heavy chain locus. , all non-human D _H gene segments, all non-human J _H gene segments, and one or more non-human C _H gene segments. In some embodiments, the 5' homology arm comprises the sequence presented as SEQ ID NO: 11 and/or the 3' homology arm comprises the sequence presented as SEQ ID NO: 12.

In some targeting vector embodiments, the targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus is the non-human Ig light chain locus. 5' and 3' homology arms targeting the non-human Ig light chain locus to include the recombinant nucleic acid molecule in an operative linkage upstream of the non-human Ig CL in, optionally wherein the non-human Ig light chain locus is The endogenous rodent Ig light chain locus and/or the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of the endogenous Ig V _L and/or J _L gene segments, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule substitutes a non-human V _L segment at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule substitutes one or more non-human V _L segments and all non-human J _L segments at the non-human Ig light chain locus. do. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule displaces all non-human V _L segments and all non-human J _H segments at the non-human Ig light chain locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus produces a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus. include In some embodiments, a targeting vector described herein is a nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and a non-human Ig light chain locus, the targeted non-human Ig light chain locus is a non-human Ig light chain locus. 5' and 3' homology arms targeting the non-human Ig light chain locus to include a recombinant nucleic acid molecule operably linked at the locus to a non-human Ig light chain regulatory sequence, optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of the endogenous Ig V _L and/or J _L gene segments, or a combination thereof, optionally Upon homologous recombination between the targeting vector and the non-human Ig light chain locus herein, the recombinant nucleic acid molecule comprises a non-human V _L segment, all non-human J _L gene segments, and a non-human CL gene at the non-human Ig light chain locus. Replace

In some targeting vector embodiments, the targeting vector is a nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus is the non-human Ig light chain 5' and 3' homology arms targeting the non-human Ig light chain κ locus to include the recombinant nucleic acid molecule in an operative linkage upstream of the non-human Ig Cκ at the κ locus, optionally wherein the non-human Ig The light chain κ locus is an endogenous rodent Ig light chain κ locus and/or the non-human Ig light chain κ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of an endogenous Ig Vκ and/or Jκ gene segment, or a combination thereof. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule replaces the non-human Vκ segment at the non-human Ig light chain κ locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule displaces one or more non-human Vκ segments and all non-human Jκ segments at the non-human Ig light chain κ locus. do. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule displaces all non-human Vκ segments and all non-human Jκ segments at the non-human Ig light chain κ locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus is operably linked to a non-human Ig light chain κ regulatory sequence at the Ig light chain κ locus. Includes recombinant nucleic acid molecules. In some targeting vector embodiments, the targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus is the Ig light chain κ locus. 5' and 3' homology arms targeting the non-human Ig light chain κ locus to include a recombinant nucleic acid molecule operably linked to a non-human Ig light chain κ regulatory sequence in the Upon homologous recombination between human Ig light chain κ loci, the recombinant nucleic acid molecule replaces the non-human Vκ segment, all non-human Jκ gene segments, and the non-human CK gene at the non-human Ig light chain κ locus.

In some targeting vector embodiments, the targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus is at the Ig light chain locus. comprising 5' and 3' homology arms targeting the non-human Ig light chain λ locus to include the recombinant nucleic acid molecule in an operative linkage upstream of the non-human Ig Cλ, optionally wherein the non-human Ig light chain λ locus is an endogenous rodent Ig light chain λ locus and/or the non-human Ig light chain λ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of an endogenous Ig Vλ and/or Jλ gene segment, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule replaces the non-human Vλ segment at the non-human Ig light chain λ locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule replaces one or more non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule displaces all non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain λ locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus is operably linked to a non-human Ig light chain λ regulatory sequence at the Ig light chain λ locus. Includes recombinant nucleic acid molecules. In some embodiments, the targeting vector is a recombinant nucleic acid molecule as described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus is at the Ig light chain locus. 5' and 3' homology arms targeting the non-human Ig light chain λ locus to include a recombinant nucleic acid molecule operably linked to a non-human Ig λ regulatory sequence. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule is a non-human Vλ segment at the non-human Ig light chain λ locus, all non-human Jλ gene segments, and a non-human Jλ gene segment at the non-human Ig light chain λ locus. -Substitute the human Cλ gene.

Also described herein is a non-human animal genome comprising a recombinant nucleic acid molecule and/or targeting vector as described herein. In some non-human animal genome examples, the non-human animal genome is a non-human animal genome. A recombinant nucleic acid molecule as described herein at an endogenous Ig locus of an animal genome, e.g., a non-human animal genome, comprises a targeting vector as described herein, wherein the targeting vector is a targeting vector at an endogenous Ig locus. Includes 5' and 3' homology arms that target. In some embodiments, the non-human animal genome is a rodent genome. In some embodiments, the non-human animal genome is a rat genome. In some embodiments, the non-human animal genome is a mouse genome.

Also described herein are non-human animals or non-human animal cells comprising recombinant nucleic acid molecules, targeting vectors, and/or non-human animal genomes as described herein. In some non-human animal embodiments, a non-human animal as described herein is a recombinant nucleic acid molecule, a targeting vector, and/or a non-human animal as described herein, in the germline, eg, in a germ cell. -comprises human animal genomes, eg, can transfer recombinant nucleic acid molecules, targeting vectors, and/or non-human animal genomes as described herein to their progeny.

Also described are methods of using a recombinant nucleic acid molecule, e.g., a targeting vector, as described herein in vitro to produce non-human cells, non-human embryos, and/or non-human animals. In some embodiments, an in vitro method of transforming an isolated cell involves introducing a recombinant nucleic acid molecule as described herein into the isolated cell, eg, by contacting the cell with a targeting vector as described herein. Include steps. In some embodiments, a cell is a host cell. In some embodiments, the cell is an embryonic stem (ES) cell. In some embodiments, a cell as described herein or a cell made according to a method described herein is a rodent cell, eg, the rodent cell is a rat cell or a mouse cell.

Also described are methods of using nucleic acid molecules, non-human cells, and/or non-human animals as described herein to make anchor-modified antigen-binding proteins. Also described are non-human animal embryos and non-human animals that may comprise and/or may develop (eg, be produced) from embryonic stem cells as described herein. Such embryos or non-human animals can be prepared by transplanting an ES cell as described herein into an embryo, and/or transplanting an embryo comprising an ES cell into a suitable host, and converting the ES cell or embryo into viable progeny. It can be developed by a method comprising maintaining the host under suitable conditions during development.

In some non-human animal embodiments as described herein (e.g., the non-human animal comprises a recombinant nucleic acid molecule, targeting vector, and/or genome as described herein and/or (generated according to the method), the non-human animal comprises (a) a comparable number of mature B cells in the spleen, (b) a comparable number of kappa positive in the spleen compared to a control non-human animal. B cells, (c) comparable numbers of lambda positive B cells in the spleen, (d) comparable levels of serum IgG and/or (e) comparable levels of serum IgM. In some embodiments, a non-human animal as described herein is capable of receiving an immune-response comparable to a control non-human animal. In some embodiments, a non-human animal as described herein each comprises an anchor and/or binds a plurality of antigens derived from a recombinant nucleic acid molecule, targeting vector, and/or non-human animal as described herein. contains protein. In some embodiments, a non-human animal as described herein further comprises a cognate receptor for a non-immunoglobulin polypeptide of interest, the receptor binding portion of which the non-immunoglobulin polypeptide of interest serves as an anchor. In some non-human animal embodiments, a non-human animal as described herein comprises a plurality of antigen-binding proteins that each specifically bind to a cognate receptor of a non-immunoglobulin polypeptide of interest, the receptor binding thereof The moiety serves as an anchor for the non-immunoglobulin polypeptide of interest.

As described herein, the non-immunoglobulin polypeptide of interest (the receptor binding moiety to which the non-immunoglobulin polypeptide of interest serves as an anchor) can include atrial natriuretic peptide (ANP). In some embodiments, the c-terminal tail of ANP (eg, NSFRY (SEQ ID NO: 3)) can function as an anchor for a cognate receptor. In some embodiments, the cognate receptor comprises a natriuretic peptide receptor (NPR), eg, NPR3, or a portion thereof.

In some non-human animal embodiments, a non-human animal as described herein is an object of interest whose receptor binding portion (eg, NSFRY (SEQ ID NO: 3)) acts as an anchor as described herein. Immunized with a cognate receptor of a non-immunoglobulin polypeptide (e.g., ANP) (optionally wherein the cognate receptor comprises a natriuretic peptide receptor (NPR), e.g., NPR3), - The human animal further comprises a plurality of antigen binding proteins that bind to cognate receptors, each comprising a KD of less than 1x10 ⁹ and/or a t½ of greater than 30 minutes. In some embodiments, at least 15% of the plurality of antigen-binding proteins can and/or block binding of a cognate receptor to a non-immunoglobulin polypeptide of interest. In some embodiments, greater than 50% of the plurality of antigen binding proteins bind cognate receptors expressed on the cell surface.

In some non-human animal embodiments described herein, the non-human animal is a rodent. In some non-human animal embodiments described herein, the non-human animal is a rat. In some non-human embodiments, the non-human animal is a mouse.

Also described are anchor-modified antigen-binding proteins encoded by nucleic acid molecules described herein or made by non-human animals described herein.

Methods of producing or obtaining nucleic acids encoding antigen-binding proteins are described herein, which methods include (i) non-human animals as described herein or prepared according to the methods described herein (e.g., e.g., a non-human animal comprising a modified immunoglobulin (Ig) variable (V) segment encoding an anchor modified Ig polypeptide) to an antigen (e.g., a cognate receptor of the non-immunoglobulin polypeptide of interest, herein of interest the receptor-binding portion of the non-immunoglobulin polypeptide on the surface of the body serves as an anchor), and (ii) eliciting an immune-response of the non-human animal to an antigen comprising an antibody that binds to the antigen or a nucleic acid encoding the same. It includes steps to bring about Some embodiments involve recovering an antigen binding protein, or nucleic acid encoding the same, from a non-human animal or non-human animal cell, eg, a B cell, and optionally fusing the B cell with a myeloma cell to form a hybridoma. It further comprises the step of forming. Some embodiments further include cloning the recovered nucleic acid into an expression construct, and optionally expressing the expression construct in a host cell. In some cloning embodiments, the method further comprises cloning the recovered nucleic acid, wherein the recovered nucleic acid encodes a human Ig constant region encoding sequence and an Ig variable domain in frame. Also described herein are B cells, hybridomas fused with B cells, or host cells expressing nucleic acids recovered from B cells. In some embodiments, the mass of each antigen binding protein identifies the presence of an anchor-modified Ig polypeptide. In some embodiments, the mass of each antigen-binding protein is determined by Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry. In some embodiments, the mass of each antigen binding protein confirms the presence of an anchor-modified Ig polypeptide, and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry.

(a) encoded by a recombinant nucleic acid molecule described herein, a targeting vector described herein, or a non-human animal genome described herein; (b) a non-human animal or non-human described herein; Anchors expressed by animal cells, (c) expressed by non-human animals or non-human animal cells prepared according to methods described herein, and/or (d) produced according to methods described herein. - Modified Ig polypeptides are described.

Other features, objects, and advantages of the non-human animals, cells, nucleic acids, and compositions disclosed herein are apparent from the detailed description of the specific examples that follow. However, while indicating specific embodiments of the present invention, it is to be understood that the detailed description is provided by way of example only and not limitation.

The drawings included herein, which consist of the following drawings, are for illustrative purposes only and are not intended to be limiting.
1 depicts illustrative, non-limiting examples of ANP-modified V _H gene segments useful as donor DNA for Cas9/GA modification, not to scale. Also shown are restriction recognition sites (XhoI, Mrel, EcoRI, AvrII, Mrel) and Spectinomycin resistance gene (SPEC). In this non-limiting example, the human V _H 1-69 gene segment is modified with a sequence encoding the C-terminal tail of atrial natriuretic protein (ANP) (NSFRY; SEQ ID NO: 3) to produce ANP-modified V _H 1- It forms donor DNA containing 69 gene segments. In general, unfilled shapes represent human sequences, filled shapes represent murine sequences, and dotted shapes represent non-human and non-murine sequences.
2 depicts an exemplary, non-limiting example of a DNA donor (represented by SEQ ID NO: 10) used to modify the V _H 1-69 gene in BAC clone VI504. The following characteristics are displayed:
- a sense DNA sequence comprising:
(a) the last seven codons of the bipartite signal peptide encoded by the germline human _VH 1-69 segment ( see split arrow labeled “signal peptide”); The nucleotide sequence encoding the entirety of the signal polypeptide is shown as SEQ ID NO: 6; The full amino acid sequence of the signal polypeptide is given as SEQ ID NO: 7,
(b) Germline Ig V _H 1-69 segment containing exon 1, intron 1 and exon 2 ( see split arrow labeled “VH” just above the DNA sequence)
(c) intron 1 of the germline Ig V _H 1-69 segment (“Intron”);
(d) a nucleotide sequence encoding the C-terminal tail of atrial natriuretic peptide (ANP) (SEQ ID NO: 3) and the G4S linker (SEQ ID NO: 5);
(e) portions of germline V _H 1-69 segments encoding FR1, CDR1 (patterned boxes), FR2, CDR2 (patterned boxes), FR3 and CDR3 (patterned boxes), and their amino acid sequences. (see divided arrow labeled IgHV1-69), and
(e) a 23-mer recombination signal sequence (RS-23 cleavage);
-Conceptual translation of the ANP-modified V _H 1-69 segment containing the leader sequence (labeled ANP-G4S-VH1-69),
-crRNA binding site used to cleave VI504 in vitro using Cas9 (labeled with 5' VH1-69 Cas9 and PAM);
- 5' and 3' overlaps used for Gibson Assembly of donor and VI504 (black lines with arrows labeled “5'VH1-69 overlap” and 3' VH1-69 overlap, respectively), and
- Restriction enzyme sites indicated by diagonal striped boxes below the sequence: EcoRI and AvrII sites were used to link the spectinomycin-resistance cassette to the donor, XhoI sites were used to remove the donor from the pUC vector backbone, and MreI sites were used to bind was used to remove the Spec cassette from the modified BAC before smooth recovery by oligo-mediated Gibson assembly.
3 shows an illustration of a non-limiting illustrative example in which the ANP-modified V _H 1-69 segment is inserted into BAC clone VI504 to generate targeting vector VI748 according to Example 1, not to scale. In general, unfilled shapes represent human sequences (e.g., unfilled triangles represent human V _H 1-69 segments, human D _H segments, and human J _H segments), and filled shapes represent murine sequences. (e.g., filled triangles represent endogenous murine _VH segments (unlabeled), filled arrows represent the murine Adam6a “a” and Adam6b “b” genes, filled ovals represent Ig enhancers, filled arrows represent murine Igμ gene “IgM”), dotted lines represent non-human and non-murine sequences (e.g., chloramphenicol resistance gene “CM”, site-specific recombination recognition site “Frt site, hygromycin resistance gene” HYG”, and spectinomycin resistance gene “SPEC”).
4 depicts a non-limiting illustrative example of insertion of a targeting vector into an immunoglobulin heavy chain variable region locus in the genome of a mouse ES cell via electroporation, and is not to scale. After electroporation, this non-limiting exemplary embodiment retains the endogenous V _H segment, shown as a filled triangle upstream of the Adam6a (“a”) gene. In general, unfilled shapes represent human sequences (e.g., unfilled triangles represent human V _H 1-69 segments, human D _H segments, and human J _H segments), and filled shapes represent murine sequences. (e.g., filled triangles represent endogenous murine _VH segments (unlabeled), filled arrows represent the murine Adam6a “a” and Adam6b “b” genes, filled ovals represent Ig enhancers, filled arrows represent murine Igμ gene “IgM”), dotted lines represent non-human and non-murine sequences (e.g., chloramphenicol resistance gene “CM”, site-specific recombination recognition site “Frt site, hygromycin resistance gene” HYG”, and spectinomycin resistance gene “SPEC”)
5A-5F show results comparing population splenocytes from ANP-VH1-69 modified mice to control VELOCIMMUNE® mice, in relation to a non-limiting example of the present invention. 5A-5B show (a) total number of cells per spleen (y-axis), CD19 ⁺ cells/spleen number (y-axis) or (b) mice transformed with ANP-modified _VH 1-69 segment ( ANP) or the percentage (Y-axis) of lymphocytes (CD19 ⁺ cells) per spleen of control VELOCIMMUNEE® animals (control) harboring humanized Ig loci. 5C to 5D show (c) total number of mature B cells (CD19 ⁺ IgD ^hi IgM ^int ) and metastatic B cells (CD19 ⁺ IgD ^int IgM ^hi ) (y-axis) or (d) ANP-modified V _H 1 Mature B cells (CD19 ⁺ IgD ^hi IgM ^int ) and metastatic B cells (CD19 ⁺ IgD ^int IgM ^hi ) as a percentage (y-axis). Figures 5e-f show (e) total number of CD19 ⁺ kappa ⁺ cells and CD19 ⁺ lambda ⁺ cells per spleen (y-axis) or (f) ANP-modified _VH 1-69 segment modified mice (ANP) or the percentages (y-axis) of CD19 ⁺ κ ⁺ cells and CD19 ⁺ λ ⁺ cells per spleen of control VELOCIMMUNE® animals (control) comprising a humanized Ig locus.
6A-6F show results comparing population bone marrow cells isolated from femurs of ANP-V _H 1-69 modified mice to control VELOCIMMUNE® mice, in accordance with non-limiting examples of the present invention. 6A-B show (a) total cell count per femur (y-axis) and CD19 ⁺ B cell count per femur (y-axis) or (b) mice transformed with ANP-modified _VH 1-69 segments ( ANP) or the percentage (y-axis) of lymphocytes (CD19 ⁺ cells) per femur of control VELOCIMMUNE® animals (control) containing humanized Ig loci. Figures 6c to d show (c) the total number of CD43 ⁺ ckit ⁺ pro-B cells per femur (y-axis) and the total number of CD43 ^- ckit ^- pre-B cells per femur (y-axis) or (d) ANP - CD43 ⁺ ckit ^{+ pro-B cells or CD43 - ckit - pre} -B cells per femur of control VELOCIMMUNE® animals (control) containing modified _VH 1-69 segments (ANP) or humanized Ig loci Gives the percentage of cells. Figures 6e-f show (e) total number of immature and mature B cells per femur (y-axis) or (f) ANP-modified _VH 1-69 segment modified mouse (ANP) or humanized Ig Percentages (y-axis) of immature and mature B cells per femur of control VELOCIMMUNE® animals (control) containing locus are presented.
Figure 7 shows serum IgG levels from ANP-V _H 1-69 modified animals and control VELOCIMMUNE® control animals analyzed by western blot analysis, in relation to a non-limiting example of the present invention.
8A-B show, with respect to a non-limiting example of the present invention, isolated from an ANP-V _H 1-69 modified animal (ANP) or a control VELOCIMMUNE® animal (control) comprising a humanized Ig locus ( Concentrations of a) serum mouse (m) IgG or (b) serum mIgM (μg/mL; y-axis) are plotted.
9 shows, in relation to a non-limiting example of the present invention, immuno-immunity against protein antigens in ANP-V _H 1-69 modified mice (ANP) or control VELOCIMMUNE® animals containing humanized Ig loci (control). A graph comparing response (antibody titer; y-axis) is shown . Of the sera tested, three serum samples were from the bleed after the third boost, while one serum sample was from the fifth boost on ANP-V _H 1-69 transformed mice. For VELOCIMMUNE® control mice, two serum samples were from bleeds after the 6th boost, while one was from the 9th boost.
10 shows, in relation to a non-limiting example of the present invention, an immune-response to a protein antigen in an ANP-V _H 1-69 modified mouse (ANP) or a control VELOCIMMUNE® animal containing a humanized Ig locus (control). (antibody titer; y-axis) . Of the sera tested, three serum samples were from the bleed after the third boost, while one serum sample was from the fifth boost on ANP-V _H 1-69 transformed mice. For VELOCIMMUNE® control mice, two serum samples were from bleeds after the 6th boost, while one was from the 9th boost.
11 shows, in relation to a non-limiting embodiment of the present invention, at 25° C. using box and whisker plots. K _D of hNPR3-MMH binding to NPR3 monoclonal antibody (mAb) isolated from an ANP-V _H 1-69 modified mouse (ANP) or a control VELOCIMMUNE® animal (control) containing a humanized Ig locus on a protein antigen. Shows a graph comparing values (y-axis).
12 shows , in relation to a non-limiting example of the present invention, a plot comprising ANP-V _H 1-69 modified mouse (ANP) or humanized Ig loci on a protein antigen at 25° C. using box and whisker plots. Graphs are shown comparing t½ values (y-axis) of hNPR3-MMH binding to NPR3 monoclonal antibody (mAb) isolated from control VELOCIMMUNE® animals (control).

Justice

The scope of the present invention is defined by the claims appended hereto, and is not limited by the specific embodiments described herein; Those skilled in the art reading this disclosure will be able to appreciate various modifications that may be equivalent to these embodiments described or otherwise fall within the scope of the claims. Generally, terms have their art-understood meanings unless otherwise specified. Explicit definitions of certain terms are provided here and below; The meaning of these and other terms in particular instances throughout this specification will be apparent to those skilled in the art from the context. Additional definitions for the following terms and other terms are presented throughout this specification. References cited in this specification, or relevant portions thereof, are incorporated herein by reference in their entirety.

The use of ordinal terms, such as “first,” “second,” “third,” etc., in a claim to modify a claim element is, in itself, any precedence, precedence, or precedence of one claim element over another. It does not indicate a sequence or temporal order in which the actions of a method are performed, but merely to distinguish one claim element having a given name from other elements having the same name (unless for the use of an ordinal term). used as a label.

Within this specification and claims, the articles “a” and “an” should be understood to include plural referents unless the context clearly dictates otherwise. A claim or description that includes an “or” between one or more members of a group indicates that one, more than one, or all group members are present in or employed in a given product or process unless indicated to the contrary or otherwise clear from the context. is or is otherwise deemed to satisfy the relevant The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise related to a given product or process. The invention also includes embodiments in which more than one, or an entire group of members, are present in, employed in, or otherwise related to a given product or process. Furthermore, the present invention is not intended to be based on one or more limitations, elements, clauses, descriptive terms, etc. from one or more recited claims unless otherwise indicated or it is clear to one skilled in the art that an objection or inconsistency may occur. It is understood to cover all variations, combinations and permutations introduced into other claims depending on which claim is dependent. Where elements are presented as a list (eg, in a Markush group or similar format), it is understood that each subgroup of such elements is also disclosed, and that any element(s) may be removed from such group. In general, when the invention, or aspect of the invention, is referred to as comprising a particular element, feature, etc., it should be understood that the embodiment or aspect of the invention consists of, or consists essentially of, that element, feature, or the like. For brevity, these embodiments are not specifically described herein in literally every case. It is also to be understood that any embodiment or aspect of the invention may be expressly excluded from the claims, regardless of whether or not that particular exclusion is recited within the specification.

As used in this application, the terms “about” and “approximately” are used equivalently. Any numerical value used in this application is meant to include any normal variation understood by one of ordinary skill in the art, with or without the use of about/approximately, for example +/- 5%.

Administration : means administering a composition to a subject or system (eg, a cell, organ, tissue, organism or related component or set of components thereof). It will be appreciated by those skilled in the art that the route of administration may vary depending on, for example, the subject or system to which the composition is being administered, the nature of the composition, the purpose of administration, and the like.

For example, in some embodiments, administration to an animal subject (eg, a human or rodent) is administered via bronchial (including bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary. , intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal instillation), transdermal, vaginal and/or vitreous may be administered. In some embodiments, administration may include intermittent administration. In some embodiments, administration can include continuous dosing (eg, perfusion) for at least a selected period of time. In some embodiments, antibodies produced by a non-human animal disclosed herein can be administered to a subject (eg, a human subject or rodent). In some embodiments, a pharmaceutical composition comprises an antibody produced by a non-human animal disclosed herein. In some embodiments, the pharmaceutical composition may include a buffer, diluent, excipient, or any combination thereof. In some embodiments, a pharmaceutical composition comprising an antibody produced by a non-human animal disclosed herein is prepared in a container, such as a vial, syringe (eg, IV syringe), or bag (eg, an IV syringe) for storage or administration. eg, an IV bag).

Affinity : refers to the strength of interaction between an antigen-binding protein and its binding partner, eg, between an antibody and a specific epitope. Antibodies that specifically bind to an epitope generally have a concentration of about 10 ^-9 M or less relative to their target epitope (eg, about 1 x 10 ^-9 M, 1 x 10 ^-10 M, 1 x 10 ^-11 M, or It has a K _D of about 1 x 10 ^-12 M). K _D is surface plasmon resonance, eg BIACORE ^™ ; It can be measured by enzyme-linked immunosorbent assay (ELISA) or other well-known methods.

Antibody : Refers to an immunoglobulin antigen-binding protein. Tetrameric antibodies comprise four polypeptide immunoglobulin (Ig) chains, eg, two Ig heavy (H) chains and two Ig light (L) chains interconnected by disulfide bonds. Each heavy chain comprises an Ig heavy chain variable domain and an Ig heavy chain constant region or domain ( _CH ). The heavy chain constant region comprises three domains, C _H 1 , C _H 2 and C _H 3 . Each Ig light chain comprises an Ig light chain variable domain and an Ig light chain constant region ( _CL ).

Ig heavy chain variable domains and Ig light chain variable domains can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each Ig heavy chain variable domain and light chain variable domain contains three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 ( Heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).

Antigen-binding proteins include immunoglobulins, antibodies, antibodies, binding proteins, etc., e.g., monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single chain Antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti-idiotype (anti-Id) antibodies, and epitope-binding of any of the foregoing refers to fragments. The terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in US Patent Application Publication No. 20070004909, incorporated herein by reference in its entirety, and US Patent Application Publication No. 20090060910 (incorporated in its entirety). Incorporated herein by reference) refers to Ig-DARTS as disclosed herein.

Biological activity : refers to the property of any agent that has activity in a biological system, in vitro or in vivo (eg, within an organism). For example, an agent that, when present in an organism, has a biological effect in the organism is considered to be biologically active.

In certain embodiments where a protein or polypeptide is biologically active, the portion of the protein or polypeptide that contributes at least one biological activity to the protein or polypeptide is commonly referred to as a “ biologically active ” portion.

Cognate : Refers to two biomolecules that normally interact (eg, a receptor and its ligand).

Comparable : A set of two or more agents, entities, situations, or conditions that may not be identical to each other, but are sufficiently similar to permit comparison between them, such that a reasonable conclusion can be drawn based on the observed differences or similarities. refers to Those skilled in the art will understand what degree of identity is necessary for two or more such agents, entities, situations, sets of conditions, etc. within a context to be considered comparable in any given situation.

Conservative : Refers to a conservative amino acid substitution, i.e., the substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein of interest, eg, the ability of the non-immunoglobulin polypeptide of interest to bind to its cognate receptor. Examples of groups of amino acids having side chains with similar chemical properties include: aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acid substitutions include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine.

In some embodiments, conservative amino acid substitutions may be substitutions of any original residue in the protein with alanine, for example as used in alanine scanning mutagenesis. In some examples, conservative substitutions with positive values are made in the PAM250 log likelihood matrix described in Gonnet, G. H. et al., 1992, Science 256:1443-1445, incorporated herein by reference in its entirety. In some embodiments, the substitution is a suitable conservative substitution, such substitution having a non-negative value in the PAM250 log-likelihood matrix.

Control : Refers to the art-understood meaning of “ control ”, a standard against which results are compared. Typically, controls are used to increase the completeness of an experiment by isolating variables to draw conclusions about them. In some embodiments, a control is a reaction or assay performed concurrently with a test reaction or assay to provide a comparator. Control ” may refer to “ control animal ”. A “ control animal ” may have a modification described herein, a modification different from that described herein, or may have no modification (ie, a wild type animal). In an experiment, a “ test ” (ie, the variable under test) is applied. In the second experiment, the " Control ," variable under test does not apply. A control can be a positive control or a negative control.

In some embodiments, a control is a historical control (ie, a control of a previously performed test or assay, or a previously known quantity or result). In some embodiments, a control is or includes a printed or otherwise stored record.

A degenerate variant of a reference nucleic acid molecule encodes a polypeptide having the same amino acid sequence as that encoded by the reference nucleic acid molecule and having substantially the same nucleic acid sequence as the reference nucleic acid molecule, but with differences due to degeneracy of the genetic code.

Disruption : Refers to the result of a homologous recombination event with DNA molecules (eg, with endogenous homologous sequences such as genes or genetic loci).

In some embodiments, a break may effect or represent an insertion, deletion, substitution, replacement, erroneous mutation, or frame-shift of a DNA sequence, or any combination thereof. Insertions can include whole genes, fragments of genes, eg, exons, which can be of origin other than endogenous sequences (eg, heterologous sequences), or insertions of coding sequences derived or isolated from a particular gene of interest. there is. In some embodiments, disruption may increase expression and/or activity of a gene or gene product (eg, a protein encoded by the gene). In some embodiments, disruption may reduce expression and/or activity of a gene or gene product. In some embodiments, a disruption may alter the sequence of a gene or an encoded gene product (eg, an encoded protein). In some embodiments, a disruption may alter the sequence of a chromosome or the location of a chromosome within a genome. In some embodiments, a disruption may cut or fragment a gene or encoded gene product (eg, encoded protein). In some embodiments, a disruption may expand a gene or encoded gene product. In some such embodiments, disruption may achieve assembly of the fusion protein. In some embodiments, a disruption may affect the level of a gene or gene product but not activity. In some embodiments, a disruption may affect the activity of a gene or gene product but not the level. In some embodiments, disruption may not have a significant effect on the level of a gene or gene product. In some embodiments, disruption may not have a significant effect on the activity of a gene or gene product. In some embodiments, disruption may have no significant effect on the level or activity of a gene or gene product. In some embodiments, a significant effect can be measured by, for example, but not limited to Student's T-test.

Endogenous Locus or Endogenous Gene : Refers to a genetic locus found in a parental or reference organism (or cell) prior to introduction of an alteration, disruption, deletion, insertion, modification, substitution or replacement described herein.

In some embodiments, an endogenous locus comprises, in whole or in part, a sequence found in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the reference organism is a wild type organism. In some embodiments, a reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-bred organism (whether wild type or engineered).

Endogenous Promoter : Refers to a promoter naturally associated with an endogenous gene or genetic locus, e.g., in a wild-type organism.

Manipulated : Generally, refers to the aspect that has been manipulated by human hands. As is common practice and is understood by those skilled in the art, a progeny of an engineered polynucleotide or cell is generally still referred to as “ engineered ” even if the actual manipulation was performed on the former entity. Additionally, as will be appreciated by those skilled in the art, a variety of methodologies are available by which “ manipulation ” as described herein can be accomplished. A polynucleotide can be considered "engineered" when two or more sequences that are not linked together in nature in that order have been engineered by human hands to link directly with each other in an engineered polynucleotide. In some embodiments, an engineered polynucleotide may include a regulatory sequence found in nature that is operably linked to a first coding sequence and not operably linked to a second coding sequence, wherein the regulatory sequence is The sequences are manually linked into operative association with the second coding sequence. Alternatively or additionally, in some embodiments, first and second nucleic acid sequences each encoding polypeptide elements or domains that are not linked to each other in nature may be linked to each other in an engineered polypeptide. In contrast, in some embodiments, a cell or organism has been engineered such that its genetic information is altered (eg, new genetic material not previously present is introduced, or previously present genetic material is altered or removed). , cells or organisms can be considered “engineered”.

In some embodiments, "manipulation" is through the use of a computer system programmed to perform analysis or comparison, or otherwise analyze, recommend and/or select sequences, alterations, etc. (e.g., nucleic acid sequences, polypeptide sequences, cells, etc.) , selecting or designing tissues and/or organisms). Alternatively or additionally, in some embodiments, "engineering" refers to in vitro chemical synthetic methodologies and/or recombination, e.g., nucleic acid amplification hybridization (via polymerase chain reaction), mutagenesis, transformation, transfection, etc. nucleic acid technology, and/or any of a variety of controlled breeding methodologies. As will be appreciated by those skilled in the art, established such methods (eg, for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection, etc.)) Various techniques are well known in the art and are described in various general and more specific references that are cited and/or discussed throughout this specification. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; incorporated herein by reference in its entirety).

Gene : Refers to a DNA sequence within a chromosome that codes for a product (eg, an RNA product and/or a polypeptide product). For clarity, as used herein, the term “gene” generally refers to a portion of a nucleic acid that encodes a polypeptide; It is noted that, as will be clear to one skilled in the art from the context, one may optionally include regulatory sequences. This definition is not intended to exclude the application of the term "gene" to non-protein-encoding expression units, but rather to clarify that, in most cases, the term refers to a polypeptide-encoding nucleic acid as used herein. will be.

In some embodiments, a gene includes a coding sequence (ie, a sequence encoding a particular product). In some embodiments, a gene comprises a non-coding sequence. In some specific embodiments, a gene may include both coding (eg, exons) and non-coding (introns) sequences. In some embodiments, a gene has one or more regulatory sequences (eg, promoters, enhancers, etc.) and/or, for example, one or more aspects of gene expression (eg, cell type specific expression, induced expression, etc.) It may contain intronic sequences that can regulate or affect.

The variable domain of an immunoglobulin antigen receptor, such as an antibody, is encoded by a set of gene segments , also referred to herein as “segments,” that are located sequentially along a chromosome and undergo somatic recombination to form complete variable domain exons. The organization of inherited human gene segments, e.g., the germline organization of human gene segments, e.g., the order of human gene segments within a human germline genome (e.g., a genome that is passed down to the next generation), is Lefranc, M. -P., Exp. Clin. Immunogenet., 18, 100-116 (2001) (the entire contents of which are incorporated herein by reference), which also describes functional gene segments and pseudogenes found within human immunoglobulin heavy chain loci in germline configurations. show A gene segment may be a variable (V) gene segment (each of which may also be individually referred to as a V segment), a diversity (D) gene segment (each of which may also be individually referred to as a D segment), or a combination ( J) gene segments (each of which may also be individually referred to as a J segment). Germline DNA contains multiple copies of each type of gene segment, but only one of each type of receptor chain is expressed in the receptor-bearing lymphocyte.

A series of recombination events involving several genetic components are responsible for assembling immunoglobulins from ordered arrangements of gene segments (eg, V, D and J). Assembly of these gene segments is known to be imprecise, so immunoglobulin diversity is achieved both by the combination of different gene segments and by the formation of unique junctions through imprecise association. Diversity is also created through a process known as somatic hypermutation in which the variable region sequences of immunoglobulins are altered to increase affinity and specificity for antigen. Reference herein to an Ig gene segment, eg, an Ig V segment, includes variants of germline Ig V segments. Variants of germline Ig segments, e.g., germline Ig V segments, include polymorphisms, e.g., alleles, variants thereof, somatically hypermutated variants thereof, recombined variants thereof, and degenerate variants thereof. .

s, N. 등의 (1998) Exp. Clin. Immunogene t., 15, 8-18; Barbi

, V. 및 Lefranc, M.-P. (1998) Exp. Clin. Immunogenet., 15, 171-183; Martinez, C. 및 Lefranc, M.-P. (1998) Exp. Clin. Immunogenet., 15, 184-193; Pallar

s, N. 등의 (1999) Exp. Clin. Immunogenet., 16, 36-60 (1999), 및 Ruiz, M. 등의 (1999) Exp. Clin. Immunogenet., 16, 173-184에 기술되어 있으며, 이들 각각은 그 전체가 참조로서 본원에 통합된다. 대립유전자 생식계열 Ig 분절의 표현은 또한 월드와이드 웹(www) 상에서 2개의 형식으로 발견될 수 있는데, imgt.org/IMGTrepertoire/Proteins/#B는 대립 유전자*01와 비교하여 상이한 대립유전자에 할당된 모든 공지된 서열의 정렬을 표시하고, imgt.org/IMGTrepertoire/Proteins/#C는 대립 유전자*01와 비교하여 뉴클레오티드 돌연변이 및 상이한 대립유전자의 상응하는 아미노산 변화에 대한 설명을 제공한다. 또한 유럽특허 제3 128 009호를 참조하고, 동 문헌은 그 전체가 참조로서 본원에 통합된다. Sequence polymorphisms in the coding regions of Ig segments, e.g., sequences of allelic variants of germline Ig segments, are Pallar

s, N. et al. (1998) Exp. Clin. Immunogen t. , 15, 8-18; Barbi

, V. and Lefranc, M.-P. (1998) Exp. Clin. Immunogenet ., 15, 171-183; Martinez, C. and Lefranc, M.-P. (1998) Exp. Clin. Immunogenet ., 15, 184-193; Pallar

s, N. et al. (1999) Exp. Clin. Immunogenet., 16, 36-60 (1999), and Ruiz, M. et al. (1999) Exp. Clin. Immunogenet. , 16, 173-184, each of which is incorporated herein by reference in its entirety. Representations of allelic germline Ig segments can also be found on the world wide web (www) in two formats: imgt.org/IMGTrepertoire/Proteins/#B assigned to different alleles compared to allele*01 Displaying an alignment of all known sequences, imgt.org/IMGTrepertoire/Proteins/#C provides a description of the nucleotide mutations and corresponding amino acid changes in the different alleles compared to allele*01. See also EP 3 128 009, incorporated herein by reference in its entirety.

An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it contains one or more of the following regions: FR1, FR2 and/or FR3, at least for germline segments 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% nucleic acid sequence homology, or at least 80%, at least 85% to an amino acid sequence encoded by a germline Ig V segment , which encode amino acids that have at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity.

In some embodiments, an Ig V segment may be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it contains one or more of the following regions: FR1, FR2 and/or FR3, germline 80% to 99% nucleic acid sequence homology to the family segment. In some embodiments, an Ig V segment may be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it contains one or more of the following regions: FR1, FR2 and/or FR3, germline 85% to 99% nucleic acid sequence homology to the family segment. In some embodiments, an Ig V segment may be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it contains one or more of the following regions: FR1, FR2 and/or FR3, germline 90% to 99% nucleic acid sequence homology to a family segment. In some embodiments, an Ig V segment may be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it contains one or more of the following regions: FR1, FR2 and/or FR3, germline 95% to 99% nucleic acid sequence homology to a family segment.

An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, by the germline Ig V segment. An amino acid having 80% to 99% amino acid sequence homology to the encoded amino acid sequence. An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, by the germline Ig V segment. An amino acid having 85% to 99% amino acid sequence homology to the encoded amino acid sequence. An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, by the germline Ig V segment. An amino acid having 90% to 99% amino acid sequence homology to the encoded amino acid sequence. An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, by the germline Ig V segment. An amino acid having 95% to 99% amino acid sequence homology to the encoded amino acid sequence.

Immunoglobulins are Y-shaped polypeptides composed of two identical heavy chains and two identical light chains, each of which has two structural components: one variable domain and one constant domain. Formed by the assembly of gene segments are the variable domains of the heavy and light chains, but the constant domains are fused to the variable domains via RNA splicing. The mechanism for assembling (or joining) gene segments is similar for heavy and light chains, but only one joining event is required for light chains (i.e. V to J) and two for heavy chains (i.e. D to J and V to DJ).

Generally, immunoglobulin heavy chain variable domains recombine with immunoglobulin heavy chain diversity (D _H ) gene segments (also referred to as D _H segments) and immunoglobulin heavy chain binding J _H gene segments (also referred to as J _H segments). It is encoded by a variable domain exon formed by somatic recombination of a modified immunoglobulin heavy chain variable (V _H ) gene segment (also referred to as a V _H segment). Immunoglobulin light chain variable domains are generally somatic recombination of an immunoglobulin light chain variable (V _L ) gene segment (also referred to as a V _L segment) and an immunoglobulin light chain joining (J _L ) gene segment (also referred to as a J _L segment). It is encoded by the variable domain exons formed by

Assembly of the gene segments for the heavy and light chain variable regions (referred to as VDJ recombination and VJ recombination, respectively) is guided by conserved noncoding DNA sequences flanking each gene segment, called recombination signal sequences (RSS) , which ensures DNA rearrangements in the correct positions relative to the V, D and J coding sequences (see, eg, Ramsden, D.A. et al., 1994, Nuc. Acids Res. 22(10):1785-96; full text thereof incorporated herein by reference). Each RSS consists of a conserved block of 7 nucleotides (7mers) contiguous with the coding sequence (e.g., V, D or J segments) followed by a conserved spacer (12bp or 23bp) and 9 nucleotides (9mers). ) of the second conserved block. Significant sequence diversity in 12 bp or 23 bp spacers between individuals is allowed, but generally the length of these sequences does not vary. Recombination between immunoglobulin gene segments follows a rule commonly referred to as the 12/23 rule, where a gene segment flanked by an RSS with a 12 bp spacer (or 12-mer) is usually a gene segment flanked by a 23 bp spacer. (or a 23-mer; see, eg, Hiom, K. and M. Gellert, 1998, Mol. Cell. 1(7):1011-9; incorporated herein by reference in its entirety).

Unless indicated otherwise, or unless it is clear to those skilled in the art that inconsistencies or inconsistencies will arise, unrearranged gene segments that do not make reference to RSS are those to which the gene segments are naturally associated, e.g. , flanked by, or operably linked to, etc. are assumed to contain two RSSs. In some embodiments, an unrearranged gene segment herein may include a gene segment of its germline (eg, wild-type) constituent, for example, a germline V _H gene segment and a germline J _H gene. A 23-mer RSS is located on each side of the segment. In contrast, germline D _H gene segments, eg, unrearranged D _H gene segments, have a 12-mer RSS located on either side of each.

As such, an unrearranged gene segment can also refer to a gene segment of a germline construct thereof, including any RSS associated with such germline construct. In addition, a plurality of gene segments of that germline construct generally refers to not only that each individual gene segment is in that germline (e.g., unrearranged) construct, but also to the order and/or location of the functional gene segments. do. For example, Lefranc, M.-P., Exp. Clin. See Immunogenet., 18, 100-116 (2001); For germline construction of human V, D and J gene segments, the entirety of which is incorporated herein by reference.

Each V segment comprises a nucleic acid sequence encoding an Ig signal or leader (L) peptide operably linked to nucleic acid sequences encoding FR1, CDR1, FR2, CDR2, FR3, and a portion of CDR3 of an immunoglobulin variable domain. . Each D gene segment contributes to CDR3 of an immunoglobulin heavy chain variable domain. Each J gene segment also contributes CDR3 and FR4 of an immunoglobulin variable domain. Amino acid positions of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 based on unique numbering are described in Lefranc et al. (2003) Dev. Comp. Immunol. 27:55-77 and also available at www.imgt.org.

Heterogeneous : Refers to agents or entities from different sources. For example, when used in reference to a polypeptide, gene, or gene product present within a particular cell or organism, the term means that the relevant polypeptide or fragment thereof, gene or fragment thereof, or gene product or fragment thereof is: (1) a human manipulated by the hands of; 2) introduced into a cell or organism (or a precursor thereof) by human hand (eg through genetic manipulation); and/or (3) is not naturally produced by or is present in a related cell or organism (eg, related cell type or organism type). Another example is by mutation or placement under the control of, for example, a non-naturally associated and, in some embodiments, a non-endogenous regulatory element (e.g., a promoter), usually present in a particular natural cell or organism, but A modified polypeptide or fragment thereof, a gene or fragment thereof, or a gene product or fragment thereof.

Host cell : refers to a cell into which a heterologous (eg, exogenous) nucleic acid or protein has been introduced. Those skilled in the art reading this disclosure will understand that these terms are used to refer not only to a particular cell of interest, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny, while not identical to the parent cell, are still included within the scope of the term "host cell".

In some embodiments, a host cell is or comprises a prokaryotic or eukaryotic cell. In some embodiments, a host cell is or comprises a mammalian cell. In general, a host cell is any cell suitable for receiving and/or producing a heterologous nucleic acid or protein, regardless of the kingdom of life to which the cell is assigned. Exemplary cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains such as Escherichia coli, Bacillus spp., Streptomyces spp.), mycobacterium Bacterial cells, fungal cells, yeast cells (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe), Pichia pastoris, Pichia methanolica ), etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas.

In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from a CHO (eg, CHOK1, DXB-11 CHO, Veggie-CHO), COS (eg, COS-7), retinal cell, Vero, CV1, kidney (eg HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (eg BHK21), Jurkat, Daudi, A431 (epithelial), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and cell lines derived from the aforementioned cells. In some embodiments, the cells include retinal cells (eg, PER.C6® cells) that express one or more viral genes, eg, viral genes. In some embodiments, a host cell is or comprises an isolated cell. In some embodiments, a host cell is part of a tissue. In some embodiments, a host cell is part of an organism.

Humanized : A molecule of non-human origin and modified (e.g., humanized) retains its biological function and/or retains a structure that performs a retained biological function, a portion of which is the equivalent of a corresponding human molecule. refers to a molecule (eg, nucleic acid, protein, etc.) that has been replaced with a part of In contrast, "human" and the like include molecules having only human origin, eg, human nucleotides or proteins comprising only human nucleotide and amino acid sequences, respectively. The term “humanized” is used to reflect that the humanized (n) molecule can be (a) a human molecule or (b) a humanized molecule.

Identity : refers to identity determined by a number of different algorithms known in the art that can be used to determine nucleotide and/or amino acid sequence identity, with respect to comparisons of sequences.

In some embodiments, the identities described herein employ ClustalW v. 1.83 (slow) alignment and Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVECTOR Inc., 2008).

Immunoglobulin refers to a class of polypeptides present in serum or expressed on the B cells of the immune system that function as antibodies, eg, antigen-binding proteins, and nucleic acids encoding the polypeptides.

A non-immunoglobulin polypeptide refers to a ligand, eg, a polypeptide, that binds to a cognate receptor . Exemplary and well-known non-immunoglobulin polypeptide:cognate receptor pairs include, but are not limited to, non-immunoglobulin polypeptides that bind, for example, to cognate G-protein coupled receptors (GPCRs). Exemplary GPCRs include, but are not limited to, chemokine receptors, glucagon receptors (eg, GLP1:GLP1R), calcitonin receptors, and melanocortin receptors. These and other cognate GPCRs, including non-immunoglobulin polypeptides that bind thereto, are well known in the art. See, for example , Wu et al. (2017) J. Mol. Biol. 429:2726-45, each of which is incorporated herein by reference in its entirety. Additional non-limiting and exemplary non-immunoglobulin polypeptide (ligand):cognate receptor pairs include:

a. Ligands that bind to cognate receptor tyrosine kinases, e.g., ligands such as epidermal growth factor (EGF), insulin, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), etc., but Not limited to this.

b. DLL: Notch Receptor Pair,

c. B7: CD28/CLTA4/PD1 receptor pair,

d. semaphorin:plexin receptor pair,

e. PCSK9/LDLR pair;

f. HLA:LILR pair,

g. HLA:KIR pair,

h. RGD-ligand:integrin pair,

i. Amylin:CALCR/RAMP, eg, RAMP1/2/3, pair

j. Natriuretic peptides (eg, ANP, BNP, CNP, etc.): natriuretic peptide receptor (NPR, NPR3, etc.) pairs, etc.

A ligand:receptor pair may include a protease and an inhibitor.

In vitro : Refers to an event that occurs not within a multicellular organism, but in an artificial environment, such as a test tube, reaction vessel, cell incubator, etc.

In vivo : Refers to events that occur within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within live cells (eg as opposed to in vitro systems).

Isolated : (1) separated from at least some of the components with which it was associated when initially created (whether in nature and/or in a laboratory setting), and/or (2) designed, produced by human hands; refers to materials and/or entities that have been fabricated and/or manufactured. Isolated substances and/or entities may be separated from about 10 or more of the other components with which they were initially associated. In some embodiments, an isolated agent is at least about 80% pure. A substance is “pure” if it is substantially free of other constituents. In some embodiments, as will be appreciated by those skilled in the art, a substance may still remain after being combined with certain other ingredients, such as, for example, one or more carriers or excipients (eg, buffers, solvents, water, etc.) can be considered “isolated” or even “pure”; In these examples, the percentage of isolation or purity of a material is calculated without including such carriers or excipients.

As an example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is: (a) associated with some or all components that accompany it in its original state in nature due to its origin or source of derivation; if there is; (b) is substantially free of other polypeptides or nucleic acids from the same species as the species that produces it in nature; or (c) is considered “isolated” if it is expressed by, or otherwise associated with, a cell or other component of an expression system that is not of the species that naturally produces it. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system that is different from the cell system that naturally produces the polypeptide is considered an “isolated” polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may comprise a) other components with which the polypeptide is naturally associated; and/or b) an "isolated" polypeptide to the extent that the polypeptide is separated from other components with which it was initially associated.

Leader sequence or signal peptide: Refers to an immunoglobulin signal or leader (L) peptide that guides the immunoglobulin heavy or light chain through the endoplasmic reticulum and is cleaved from the heavy or light chain before the final antibody is assembled. It can also refer to a nucleic acid sequence encoding a signal or leader peptide. Each V gene segment is composed of exon 1 and exon 2 of the segment immediately upstream of the exon 2 sequence encoding FR1, CDR1, FR2, CDR2, FR3 and CDR3 of the germline Ig V segment ( eg , see FIG . 2 ). It includes a leader sequence encoded by Ig signals or leader sequences are well known in the art. For example , Lefranc et al. (2003) Dev. Comp. Immunol. 27:55-77, which is incorporated herein by reference in its entirety, and is also available on the world wide web (www) at imgt.org. See also Biomedicines 8(9):1-117 by Lefranc and Lefranc (2020).

Non-Human Animal : Refers to any vertebrate organism that is not human. Non-human animals can be protozoans, bony fish, cartilaginous fish (eg, sharks or rays), amphibians, reptiles, mammals, and birds. In some embodiments, a non-human mammal may be a primate, goat, sheep, pig, dog, cow, or rodent. In some embodiments, a non-human animal may be a rat or mouse.

Nucleic acid , in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain and is generally interchangeable with a nucleic acid molecule, nucleic acid sequence, nucleotide molecule, nucleotide molecule, and these terms also refer to each other. are interchangeable

In some embodiments, a “nucleic acid” is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from the context, in some embodiments “nucleic acid” refers to individual nucleic acid residues (eg, nucleotides and/or nucleosides); In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” is or comprises RNA; In some embodiments, “nucleic acid” is or comprises DNA. In some embodiments, a “nucleic acid” is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a “nucleic acid” is, comprises, or consists of one or more nucleic acid analogues. In some embodiments, nucleic acid analogs differ from “nucleic acids” in that they do not utilize a phosphodiester backbone. For example, in some embodiments, a “nucleic acid” is one or more “peptide nucleic acids” that are known in the art, have peptide bonds in place of phosphodiester bonds in the backbone, and are considered within the scope of this invention. (peptide nucleic acid)” is, includes, or consists of. Alternatively or additionally, in some embodiments, a “nucleic acid” has one or more phosphorothioate and/or 5′-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a “nucleic acid” is one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), comprising, or consisting of. In some embodiments, “nucleic acid” refers to one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uri Deine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-dezaadenosine, 7-dezaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methyl guanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), comprises, or consists of. In some embodiments, “nucleic acid” includes one or more modified sugars compared to the sugars of natural nucleic acids (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). do. In some embodiments, “nucleic acid” has a nucleotide sequence that encodes a functional gene product such as RNA or protein. In some embodiments, a “nucleic acid” has a nucleotide sequence that encodes a polypeptide fragment (eg, a peptide). In some embodiments, a “nucleic acid” includes one or more introns. In some embodiments, a “nucleic acid” includes one or more exons. In some embodiments, “nucleic acid” includes one or more coding sequences. In some embodiments, a "nucleic acid" is one of one or more isolates from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on complementary templates, reproduction in a recombinant cell or system, and chemical synthesis. Manufactured by the above. In some embodiments, a “nucleic acid” is at least 3 or more residues in length. In some embodiments, “nucleic acid” is single stranded; In some embodiments, “nucleic acid” is double-stranded. In some embodiments, a “nucleic acid” has a nucleotide sequence that is complementary to, or comprises at least one element that encodes, a sequence that encodes a polypeptide or fragment thereof. In some embodiments, a “nucleic acid” has enzymatic activity.

Operably linked : refers to the juxtaposition in which the described components are in a relationship that allows them to function in their intended manner.

In other embodiments, an operative connection does not require adjacency. For example, unrearranged variable region gene segments that are "operably linked" to each other can be rearranged to form a rearranged variable region gene, which means that the unrearranged variable region gene segments must be adjacent to each other. There is no need. Unrearranged variable region gene segments operably linked to each other and to adjacent constant region genes can be rearranged to form a rearranged variable region gene that is expressed together with the constant region gene as a polypeptide chain of an antigen binding protein. . Control sequences " operably linked " to a coding sequence are linked such that expression of the coding sequence is achieved under conditions compatible with the control sequences. expression control sequences that act at a distance or in trans for

The term “ expression control sequence ” refers to polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are linked. “ Expression control sequences ” include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translational efficiency; sequences that promote protein stability (ie, Kozak consensus sequences); and, if desired, sequences that enhance protein secretion. The nature of these regulatory sequences varies depending on the host organism. For example, in prokaryotes, such regulatory sequences usually include a promoter, ribosome binding site, and transcription termination sequences, whereas in eukaryotes, such regulatory sequences usually include promoters and transcription termination sequences. The term " control sequence " is intended to include components whose presence is essential for expression and processing, and may also include additional components which would be advantageous to be present, such as leader sequences and fusion partner sequences.

Physiological condition : has the meaning understood in that field to refer to the condition in which a cell or organism lives and/or reproduces. In some embodiments, the term refers to conditions in the external or internal environment that may occur in nature for an organism or cell system. In some embodiments, a physiological condition is a condition that exists within the body of a human or non-human animal, particularly conditions that exist at and/or within a surgical site. Physiological conditions are typically, for example, the temperature range of 20-40° C., 1 atmospheric pressure, pH 6-8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric level, and gravity as they are encountered on the ground. include In some embodiments, conditions in the laboratory are manipulated and/or maintained at physiological conditions. In some embodiments, physiological conditions are encountered in organisms (eg, non-human animals).

Polypeptide : refers to any polymeric chain of amino acids.

In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that contains parts that occur in nature separately from each other (ie, from two or more different organisms, eg, human and non-human parts). In some embodiments, a polypeptide has an engineered amino acid sequence designed and/or produced through the work of man. In some embodiments, a polypeptide may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., Fragments that are directly linked to the parent may be spatially separated from the polypeptide of interest or vice versa, and/or the fragments may be in a different order within the polypeptide of interest than in the parent), the polypeptide of interest may have its parent Become a derivative of a polypeptide.

Recombinant : Polypeptides expressed using recombinant means, such as recombinant expression vectors transfected into host cells, polypeptides isolated from recombinant, combinatorial human polypeptide libraries (Hoogenboom HR, 1997 TIB Tech. 15:62-70; Hoogenboom H., and Chames P., 2000, Immunology Today 21:371-378; Azzazy H., and Highsmith WE, 2002, Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW, 2002, BioTechniques 29:128-145); Antibodies isolated from animals (eg, mice) transgenic for human immunoglobulin genes (eg, Taylor, LD, et al., 1992, Nucl. Acids Res. 20:6287-6295; Little M. et al., 2000, Immunology Today 21:364-370;Kellermann SA and Green LL, 2002, Current Opinion in Biotechnology 13:593-597;Murphy, AJ, et al., 2014, Proc. Natl. Acad. Sci. USA 111(14): 5153-5158; each of which is incorporated herein by reference in its entirety), or any other means comprising splicing selected sequence elements to each other. refers to a polypeptide made, expressed, produced or isolated by

In some embodiments, one or more of these selected sequence elements are found in nature. In some embodiments, one or more of these selected sequence elements are designed in silico . In some embodiments, one or more such selected sequence elements are derived from mutagenesis (eg, in vivo or in vitro ) of known sequence elements, eg, from natural or synthetic sources. For example, in some embodiments, a recombinant polypeptide comprises a sequence found in the genome (eg, polypeptide) of a source organism of interest (eg, human, mouse, etc.). In some embodiments, recombinant polypeptides occur in nature in two different organisms (eg, a human and a non-human organism) separately from each other (ie, from two or more different organisms, eg, a human and a non-human site). contains a sequence that In some embodiments, a recombinant polypeptide has an amino acid sequence derived from mutagenesis (eg, in a non-human animal, eg, in vitro or in vivo ) such that the amino acid sequence of the recombinant polypeptide is derived from the polypeptide sequence. originated and related to it, but in vivo in non-human animals. A sequence that may not exist naturally within the genome.

Reference : Refers to a standard agent or control agent, animal, cohort, subject, population, sample, sequence or value to which an agent, animal, cohort, subject, population, sample, sequence of interest or value of interest is compared. A “ reference ” or “ control” may refer to a “ reference animal ” or a “ control animal ”. A “ reference animal ” may have a strain described herein, a strain different from that described herein, or no strain (ie, a wild type animal). Typically, as will be understood by one skilled in the art, a reference agent, animal, cohort, individual, population, sample, sequence or value is an agent of interest, animal (e.g., mammal), cohort, determined or characterized under conditions comparable to those used to determine or characterize the individual, population, sample, sequence or value.

In some embodiments, a reference agent, animal, cohort, subject, population, sample, sequence or value is tested and/or tested substantially simultaneously with the testing or determination of the agent, animal, cohort, subject, population, sample, sequence or value of interest. or determined In some embodiments, a reference agent, animal, cohort, individual, population, sample, sequence or value is a historical reference, optionally embodied in a tangible medium. In some embodiments, a criterion may refer to a control group. “VELOCIMMUNE® control” and the like, eg, “control VELOCIMMUNE®” as a reference animal or “control” refers to a VELOCIMMUNE® mouse comprising a humanized heavy chain and kappa variable region locus at which the mouse is capable of breeding. These VELOCIMMUNE® control mice are generally described in Macdonald et al. (2014) Proc. Natl. Acad. Sci. USA 111:5147-52 and supplemental information, which is incorporated herein by reference in its entirety.

Somatic Recombination: Refers to recombination of the V _H , D _H , and J _H gene segments at the immunoglobulin heavy chain locus or the V _L and J _L gene segments at the immunoglobulin light chain locus. Somatic recombination occurs before antigen contact ad during B cell development in the bone marrow. At the heavy chain locus, one D _H and one J _H recombine randomly with removal of all intervening DNA in a process termed DJ linkage. Next, the random V _H segments recombine into the rearranged D _H J _H segments. Recombination at immunoglobulin light chain loci occurs in a similar manner. The V _L gene segments and the J _L gene segments are combined and recombined in a process called VJ joining, and all DNA is removed between them.

Substantially : Includes qualitative terms that indicate a full range or degree, or a range or degree close to the whole, of the property or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely complete and/or reach completion, achieve or avoid absolute results. Accordingly, the term “ substantially ” is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Substantial homology : Refers to a comparison between amino acid or nucleic acid sequences. As understood by one skilled in the art, two sequences are generally considered to be “ substantially homologous ” if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be unequal residues that have suitably similar structural and/or functional properties. For example, as known to those skilled in the art, certain amino acids are generally classified as “ hydrophobic ” or “ hydrophilic ” amino acids, and/or as having “ polar ” or “ non-polar ” side chains. Substitution of one amino acid for another of the same type can often be considered a “ homologous ” substitution. A general classification of amino acids is summarized below.

As is known in the art, amino acid or nucleic acid sequences can be synthesized by any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP for amino acid sequences, gap BLAST and PSI-BLAST. can be compared using Exemplary such programs are described in Altschul, S. F. et al., 1990, J. Mol. Biol., 215(3): 403-410; Altschul, S. F. et al., 1996, Methods in Enzymol. 266:460-80; Altschul, S. F. et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, A.D., and B. F. F. Ouellette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1998. In addition to identifying homologous sequences, the programs described above generally provide an indication of the degree of homology.

In some embodiments, two sequences are considered substantially homologous if at least 95% or more of the corresponding residues are homologous over the relevant span of residues. In some embodiments, a relevant segment is a complete sequence. In some embodiments, the relevant interval is at least 9 or more residues. In some embodiments, the relevant interval includes contiguous residues along the complete sequence. In some embodiments, the relevant interval includes discontinuous residues along the entire sequence, eg, noncontiguous residues brought together by a folded conformation of a polypeptide or portion thereof. In some embodiments, the relevant interval is at least 10 or more residues.

Substantial identity : refers to a comparison between amino acid or nucleic acid sequences. As understood by one skilled in the art, two sequences are generally considered “ substantially identical ” if they contain identical residues at corresponding positions. As is known in the art, amino acid or nucleic acid sequences can be synthesized by any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP for amino acid sequences, gap BLAST and PSI-BLAST. can be compared using Exemplary such programs are described in Altschul, SF et al., 1990, J. Mol. Biol., 215(3): 403-410; Altschul, SF et al., 1996, Methods in Enzymol. 266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-3402; Baxevanis, AD, and BFF Ouellette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1998. In addition to identifying sequences that are identical, the programs described above generally provide an indication of the degree of identity.

In some embodiments, two sequences are considered substantially identical if at least 95% or more of the corresponding residues are identical over the relevant span of residues. In some embodiments, a relevant segment is a complete sequence. In some embodiments, the relevant interval is at least 10 or more residues.

Targeting vector or targeting construct : refers to a polynucleotide molecule comprising a targeting region. A targeting region comprises a sequence that is identical or substantially identical to a sequence in a target cell, tissue or animal and which provides integration of the targeting construct to a location in the genome of a cell, tissue or animal via homologous recombination. Targeting regions that are targeted using site-specific recombinase recognition sites (eg, lox P or Frt sites) are also included.

In some embodiments, targeting constructs described herein are mediated through the exogenous addition of specific nucleic acid sequences or genes of interest, selectable markers, control and/or regulatory sequences, and proteins that assist or promote recombination comprising such sequences. It further includes other nucleic acid sequences that allow recombination to occur. In some embodiments, a targeting construct as described herein further comprises, in whole or in part, a gene of interest, wherein the gene of interest encodes in whole or in part a polypeptide having a similar function to a protein encoded by the endogenous sequence. It is a gene. In some embodiments, a targeting construct as described herein further comprises in whole or in part a humanized gene of interest, wherein the humanized gene of interest encodes in whole or in part a polypeptide having a similar function to a polypeptide encoded by the endogenous sequence. do. In some embodiments, a targeting construct (or targeting vector) may include a nucleic acid sequence that has been manipulated by humans. For example, in some embodiments, a targeting construct (or targeting vector) is two or more humanly engineered polynucleotides or recombinant polynucleotides that are not linked together in that order in nature but are directly linked to each other in an engineered or recombinant polynucleotide. It can be constructed to contain engineered polynucleotides containing sequences or recombinant polynucleotides.

Transgene or transgene construct : Refers to a nucleic acid sequence (eg, encoding, in whole or in part, a polypeptide of interest) introduced into a cell by the human hand, such as by the methods described herein. In addition, the transgene may be partially or wholly heterologous, i.e., foreign to the transgenic animal or cell into which the transgene is introduced. A transgene may include one or more transcriptional regulatory sequences and any other nucleic acids that may be necessary for expression of the selected nucleic acid sequence, such as introns or promoters. A transgene may include one or more selectable markers that permit subsequent selection of progeny (eg, cells) that have taken up the transgene.

Transgenic animal , transgenic non-human animal, or Tg ⁺ : any non-naturally occurring ratio in which one or more of the cells of the non-human animal contain, in whole or in part, a heterologous nucleic acid and/or gene encoding a polypeptide of interest. -Can be used interchangeably with or refers to human animals.

In some embodiments, the heterologous nucleic acid sequence and/or gene is introduced directly or indirectly into a cell by introducing it into a precursor cell by deliberate genetic manipulation, such as by microinjection or infection with a recombinant virus. The term genetic manipulation does not include classical breeding techniques, but rather relates to the introduction of recombinant DNA molecule(s). These molecules can integrate into a chromosome or replicate DNA extrachromosomally. The term “ Tg ⁺ ” includes animals that are heterozygous or homozygous for a heterologous nucleic acid and/or gene, and/or that have single or multiple copies of the heterologous nucleic acid and/or gene.

Targeting vector : Refers to a nucleic acid molecule capable of transporting a nucleic acid with which a nucleic acid of interest is associated, in particular for targeting and inserting a nucleic acid of interest into another nucleic acid molecule, eg, a donor plasmid, non-human animal genome, etc.

In some embodiments, vectors are capable of extrachromosomal replication and/or expression of nucleic acids to which they are linked in a host cell, such as a eukaryotic cell and/or a prokaryotic cell. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as “ expression vectors ” or “ constructs ”.

Wild type : has the meaning understood in the art, referring to an entity that has the structure and/or activity found in nature in its “ normal ” state (as opposed to mutation, disease, alteration, etc.). Those skilled in the art will understand that wild-type genes and polypeptides often exist in many different forms (eg, alleles).

Other features, objects and advantages of the present invention will become apparent from the detailed description of specific embodiments that follow. It should be understood, however, that the detailed description, while indicating some embodiments of the invention, is provided by way of example only and not limitation. Various modifications and variations within the scope of the invention will become apparent to those skilled in the art from the detailed description.

Specific details for carrying out the invention

Antibody-based therapeutics offer significant promise in the treatment of several diseases, but the development of particularly effective antibody formulations that bind to refractory targets remains a challenge. Described herein are anchor-modified immunoglobulins (Igs) and modified Ig V segments that encode them, wherein the anchors bind at least a receptor-binding portion of a ligand (e.g., a non-immunoglobulin polypeptide) that binds a cognate receptor. include Animals harboring modified Ig V segments can produce a diverse repertoire of anchor-modified immunoglobulins in response to immunization against an anchor with a receptor homologue.

Without wishing to be bound by theory, an anchor can be, for example, a parent of an antibody that does not normally expand without the anchor by simultaneously binding to the cognate receptor with an antibody that specifically binds to the cognate receptor and/or in response to antigen challenge to the cognate receptor. By allowing chemotactic maturation, it is believed to serve to increase the affinity of newly generated antibodies in animals in response to antigenic challenge to their cognate receptors. Thus, the diverse repertoire of immunoglobulins produced by non-human animals disclosed herein may include anchor-modified immunoglobulins that bind cognate receptors with high affinity, and thus lead candidates for previously refractory targets. can increase the population of immunoglobulins that can be found.

Nucleic acid molecules including targeting vectors and animal genomes, etc.

The anchor-modified immunoglobulins described herein are at least partially by variable region (V) segments, e.g., immunoglobulin (Ig) heavy chain variable region (V _H ) segments or Ig light chain variable region (V _L ) segments. can be encoded and modified to encode anchors that are operably linked to and between the Ig leader sequence and the framework (FR) and complementarity determining regions (CDRs) of the germline Ig V segment. Nucleic acid sequences encoding immunoglobulin (Ig) signal peptides and nucleic acid sequences of germline V segments, eg, human germline V segments, are well known in the art, as are the amino acid sequences so encoded. For example, Lefranc, M.-P., Exp. Clin. Immunogenet ., 18, 100-116 (2001), which is incorporated herein by reference in its entirety, and can be found on the World Wide Web (www) at imgt.org.kr.

Nucleic acid molecules, including targeting vectors, and animal genomes described herein may include nucleic acid sequences encoding any Ig signal peptide of a germline V segment. In some embodiments, the modified Ig V segment comprises a nucleic acid sequence encoding a signal peptide of a first germline Ig V segment, a nucleic acid sequence encoding an anchor, and a nucleic acid sequence encoding framework region (FR) 1, a second Complementarity determining regions (CDRs) 1, FR2, CDR2, FR3, and CDR3 of germline Ig V segments, wherein the first germline Ig V segment and the second germline Ig segment are different germline Ig V segments. In some embodiments, the modified Ig V segment comprises a nucleic acid sequence encoding a signal peptide of a first germline Ig V segment, a nucleic acid sequence encoding an anchor, and a framework region (FR) 1 of a second germline Ig V segment. , complementarity determining regions (CDRs) 1, FR2, CDR2, FR3, and nucleic acids encoding CDR3, wherein the first germline Ig V segment and the second germline V segment are the same germline V segment. In some embodiments, the Ig signal peptide comprises the sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7).

Human (h) germline V segments (eg, human germline variable heavy chain (hV _H or hIGVH) segments, human germline variable kappa (hVκ or hIGKV) segments, and human germline variable lambda (hVλ or hIGLV) segments) and a murine (m) germline V segment (e.g., a mouse germline variable heavy chain (mV _H or mIGVH) segment, a mouse germline variable kappa (mVκ or mIGKV) segment, and a mouse germline variable lambda (mVλ or mIGLV) segment). The amino acid sequences of the fragments can be found on the world wide web at the addresses imgt.org/IMGTrepertoire/Proteins/SequenceLogos/human/ and imgt.org/IMGTrepertoire/Proteins/SequenceLogos/mouse/, each of which is incorporated herein by reference in its entirety. are integrated

In some embodiments, a germline Ig V segment or variant thereof (eg, encoding FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a modified Ig V segment as described herein) is a human (h ) germline Ig V segment or variant thereof, eg, germline human (h) V _H 1-2 segment, germline hV _H 1-3 segment, germline hV _H 1-8 segment, germline hV _H 1 18 segments, germline hV _H 1-24 segments, germline hV _H 1-45 segments, germline hV _H 1-46 segments, germline hV _H 1-58 segments, germline hV _H 1-69 segments, germline hV _H 2-5 segments, germline hV _H 2-26 segments, germline hV _H 2 segments 70, germline hV _H 3-7 segments, germline hV _H 3-9 segments, germline hV _H 3 segments 11, germline hV _H 3 13 segments, germline hV _H 3-15 segments, germline hV _H 3-16 segments, germline hV _H 3-20 segments, germline hV _H 3-21 segments, germline hV _H 3- 23 segments, germline hV _H 3-30 segments, germline hV _H 3-30-3 segments, germline hV _H 3-30-5 segments, germline hV _H 3-33 segments, germline hV _H 3-35 segment, germline hV _H 3-38 segment, germline hV _H 3-43 segment, germline hV _H 3-48 segment, germline hV _H 3-49 segment, germline hV _H 3-53 segment, germline hV _H 3-64 segments, germline hV _H 3-66 segments, germline hV _H 3-72 segments, germline hV _H 3-73 segments, germline hV _H 3-74 segments, germline hV _H 4-4 segments , germline hV _H 4-28 segment, germline hV _H 4-30-1 segment, germline hV _H 4 30-2 segment, germline hV _H 4-30-4 segment, germline hV _H 4-31 segment , germline hV _H 4-34 segment, germline hV _H 4-39 segment, germline hV _H 4-59 segment, germline hV _H 4-61 segment, germline hV _H 5-51 segment, germline hV _H 6-1 segment, germline hV _H 7-4-1 segment, germline hV _H 7-81 segment, or a variant thereof. In some embodiments, the germline Ig V segment or variant thereof is a germline hV _H 1-69 segment or variant thereof.

In some embodiments, a nucleic acid molecule as described herein comprises only a modified Ig hV _H segment, eg, no additional hV _H segments or variants thereof.

In some embodiments, a nucleic acid molecule as described herein, besides a modified Ig hV _H segment, may include additional hV _H segments, e.g., V _H 1-2, hV _H 1-3, hV _H 1-8, hV _H 1 18, hV _H 1-24, hV _H 1-45, hV _H 1-46, hV _H 1-58, hV _H 1-69, hV _H 2-5, hV _H 2-26, hV _H 2 70, hV _H 3-7, hV _H 3-9, hV _H 3 11, hV _H 3 13, hV H 3-15, hV _H 3-16, hV _H 3-20, _{hV H 3-21} , hV _H 3-23, hV _H 3-30, hV _H 3-30-3, _{hV H} 3-30-5, hV _H 3-33, hV H 3-35, hV _H 3-38, hV _H 3-43, hV _H 3-48, hV _H 3-49, hV _H 3-53, hV _H 3-64, hV _H 3-66, _hV H 3-72, hV _H 3-73, hV _H 3-74, hV _H 4-4, hV _H 4-28, hV _H 4-30-1, hV _H 4 30-2, hV _{H 4-30-4, hV H} 4-31, hV _H 4-34, _{hV H 4-39} , hV _H 4-59, hV _H 4-61, hV _H 5-51, hV _H 6-1, hV _H 7-4-1, hV _H 7-81 and variants thereof, more than one of these, or further comprising each of these. V _H 1-2, hV _H 1-3, hV _H 1-8, hV _H 1 18, hV _H 1-24, hV _{H 1-45, hV H} 1-46, _{hV H 1-58} , hV _H 1 -69, hV _H 2-5, hV _H 2-26, hV _H 2 70, hV _H 3-7, hV _H 3-9, hV H 3 11, hV _H 3 13, _{hV H 3-15} , hV _H 3-16, hV _H 3-20, hV _H 3-21, hV _H 3-23, hV _H 3-30, hV _H 3-30-3, hV _H 3-30-5, hV _H 3-33, hV _H 3-35, hV _H 3-38, hV _H 3-43, hV _H 3-48, hV _H 3-49, hV H 3-53, hV _H 3-64, _{hV H 3-66} , hV _H 3-72, hV _H 3-73, hV _H 3-74, hV _H 4-4, hV _H 4-28, hV _H 4-30-1, hV _H 4 30-2, hV _H 4-30-4 , hV _H 4-31, hV _H 4-34, hV _H 4-39, hV _H 4-59, hV _H 4-61, hV _H 5-51, hV _H 6-1, hV _H 7-4-1 , and hV _H 7-81 segments, or in some embodiments that include each of these, the hV _H segments are in germline configuration.

In some embodiments, a nucleic acid molecule as described herein (eg, a targeting vector, a non-human animal genome, etc.) may include an Ig heavy chain variable region, eg, at an anchor-modified V segment. In addition, it may contain additional rearranged (non-rearranged) V _H , D _H and/or J _H gene segments, and in some embodiments, additional rearranged (un-rearranged) hV _H , hD _H and / or the hJ _H gene segment. In some embodiments, a nucleic acid molecule as described herein (eg, a targeting vector, a non-human animal genome, etc.) has only one rearranged (non-rearranged) hV H segment, one or more rearranged (non-rearranged) hV _H segments. A nucleic acid sequence comprising an unrearranged (unrearranged) hD _H segment, and one or more rearranged (unrearranged) hJ _H segments, wherein only one rearranged (unrearranged) hV _H segment encodes an anchor as described herein. It is a modified hV _H segment that includes.

In some embodiments, a nucleic acid molecule as described herein (eg, a targeting vector, a non-human animal genome, etc.) comprises one or more human D _H segments, eg, hD _H 1-1, hD _H 1- 7, hDH _1-14 , hDH 1-20, hDH _1-26 , hDH 2-2, _{hDH 2-8} , _hDH 2-15, _hDH 2-21, _hDH 3-3 _, hD _H 3-9, hD _H 3-10, hD _H 3-16, hD _H 3-22, hD _H 4-4, hD H 4-11, hD _H 4-17, hD _H 4-23, _{hD H} 5-5, hDH _5-12 , _hDH 5-18, _hDH 5-24, hDH 6-6, _hDH 6-13, _hDH 6-19 _{, hDH} 6-25, hDH _7- 27, and one, more than one or each of their variants. hD _H 1-1, hD _H 1-7, hD _H 1-14, hD _H 1-20, hD _H 1-26, hD H 2-2, hD _H 2-8, hD _H 2-15, _{hD H} 2-21, hD _H 3-3, hD _H 3-9, hD _H 3-10, hD _H 3-16, hD _H 3-22, hD _H 4-4, hD _H 4-11, hD _H 4- 17, hDH _4-23 , hDH 5-5, _{hDH 5-12} , hDH 5-18, _hDH 5-24, _hDH 6-6 _{, hDH} 6-13, _hDH 6-19, In some embodiments comprising more than one or each of hD _H 6-25, and hD _H 7-27, the hD _H segment is in germline configuration.

In some embodiments, a nucleic acid molecule as described herein (eg, a targeting vector, a non-human animal genome, etc.) comprises one or more human J _H segments, eg, hJ _H 1, hJ _H 2, hJ _H 3, hJ _H 4, hJ _H 5, hJ _H 6, and variants thereof, more than one, or each of these. In some embodiments comprising more than one or each of hJ _H 1 , hJ _H 2 , hJ _H 3 , hJ _H 4 , hJ _H 5 , and hJ _H 6 segments, the hJ _H segment is in germline configuration. .

In some embodiments, a nucleic acid molecule as described herein (e.g., a targeting vector, a non-human animal genome, etc.) may include a heavy chain variable region locus, e.g., 5' to 3 in operative linkage. ', which may include: (I) a modified Ig V _H segment, (II) one or multiple Ig heavy chain diversity (D _H ) segments, and (III) one or multiple all Ig heavy chain bonds (J _{H )} ) segmentation. In some embodiments, the one or plurality of Ig D _H segments of (II) comprises one, plurality, or all human Ig D _H segments, and/or the one or plurality of Ig J _H segments of (III) comprises one , multiple, or all human Ig J _H segments. In some embodiments, the one or plurality of Ig D _H gene segments of (II) and the one or plurality of Ig J _H gene segments of (III), wherein the recombinant nucleic acid molecule is a modified Ig V _H gene segment and a rearranged Ig D gene segment. Recombined to include the _H /J _H sequence 5' to 3' in an operative linkage to form a rearranged Ig D _H /J _H sequence.

In some embodiments, the Ig V _H gene segment and the rearranged Ig D _H /J _H sequence are recombined to form a rearranged Ig V _H /D _H /J _H sequence encoding an anchor modified Ig heavy chain variable domain; , wherein the anchor modified Ig heavy chain variable domain comprises FR1, a complementarity determining region (CDR) encoded by (i) an Ig signal peptide, (ii) an anchor, and (iii) a rearranged Ig V _H /D _H /J _H sequence. ) 1, FR2, CDR2, FR3, CDR3, and FR4 as operative linkages.

In some embodiments, the modified Ig V _H segment is an unrearranged modified Ig V _H gene segment.

In some embodiments, a recombinant nucleic acid (eg, targeting vector, non-human animal genome, etc.) as described herein further comprises a nucleic acid sequence encoding an Ig heavy chain constant region ( _CH ), wherein the Ig The nucleic acid sequence encoding C _H is operably linked downstream of (I) a modified Ig V _H segment, (II) one or a plurality of Ig D _H segments, and (III) one or a plurality of Ig J _H segments. . In some embodiments, the nucleic acid sequence encoding Ig _CH is an Igμ gene encoding an IgM isotype, an Igδ gene encoding an IgD isotype, an Igγ gene encoding an IgG isotype, an Igα gene encoding an IgA isotype, and/or an Igε gene encoding an IgE isoform. In some embodiments, a recombinant nucleic acid molecule described herein comprises a nucleic acid sequence encoding an anchor-modified Ig heavy chain, wherein the anchor-modified Ig heavy chain comprises (i) an Ig signal peptide, (ii) an anchor, (iii) ) an Ig heavy chain variable domain comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _H /D _H /J _H sequence, and (iv) an Ig C _H operable include in connection In some embodiments, the Ig _CH is a non-human Ig _CH , eg, a rodent Ig _CH , eg, a rat Ig _CH or a mouse Ig _CH .

In some embodiments, a germline Ig V segment or variant thereof (e.g., one encoding FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a modified Ig V segment as described herein) is germline Ig light chain variable (V _L ) segments or variants thereof. In some embodiments, a recombinant nucleic acid molecule may comprise a light chain variable region locus, e.g., in an operative linkage and 5' to 3': (I) a modified Ig V _L segment , and (II) one or multiple Ig light chain linkage (J _L ) segments. In some embodiments, the modified Ig V _L segment and one or plurality of Ig J _L segments are recombined to form a rearranged Ig V _L /J _L sequence encoding an anchor modified Ig light chain variable domain, wherein the anchor modification The modified Ig light chain variable domain comprises FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by (i) an Ig signal peptide, (ii) an anchor, and (iii) a rearranged Ig V _L /J _L sequence. includes as an operable link. In some embodiments, a recombinant nucleic acid molecule may comprise a nucleic acid sequence encoding a light chain variable region locus and an Ig light chain constant region ( _CL ), wherein the nucleic acid sequence encoding an Ig _CL is downstream and operable (I) modified Ig V _L segments and (II) one or multiple Ig light chain linking (J _L ) segments. In some embodiments, an anchor-modified Ig light chain comprises (i) an Ig signal peptide, (ii) an anchor, (iii) FR1, CDR1, FR2, CDR2, FR3 encoded by a rearranged Ig V _L /J _L sequence. , an Ig light chain variable domain comprising CDR3, and FR4, and (iv) an Ig C _L in operable linkage. In some embodiments, the Ig _CL is a non-human Ig _CL , eg, a rodent Ig _CL , eg, a rat Ig _CL or a mouse Ig _CL .

In some embodiments, a germline Ig V segment or variant thereof (eg, encoding FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a modified Ig V segment as described herein) is a germline Ig light chain variable kappa (Vκ) segments or variants thereof, e.g., human Vκ segments, e.g., hVκ1-5 segments, hVκ 1-6 segments, hVκ1-8 segments, hVκ1D-8 segments, hVκ1-9 segments , hVκ1-12 segment, hVκ1D-12 segment, hVκ1-13 segment, hVκ1D-13 segment, hVκ1-16 segment, hVκ1D-16 segment, hVκ1-17 segment, hVκ1D-17 segment, hVκ1-27 segment, hVκ1-33 segment , hVκ1D-33 segment, hVκ1-37 segment, hVκ1D-37 segment, hVκ1-39 segment, hVκ1D-39, a hVκ1-NL1 segment, hVκ1D-42 segment, hVκ1D-43 segment, hVκ2-4 segment, hVκ2-18 segment , hVκ2D-18 segment, hVκ2-24 segment, hVκ2D-24 segment, hVκ2-28 segment, hVκ2D-28 segment, hVκ2-29 segment, hVκ2D-29 segment, hVκ2-30 segment, hVκ2D-30 segment, hVκ2-40 segment , hVκ2D-40 segment, hVκ2D-26 segment, hVκ3-7 segment, hVκ3D-7 segment, hVκ3-11 segment, hVκ3D-11 segment, hVκ3-15 segment, hVκ3D-15 segment, hVκ3-20 segment, hVκ3D-20 segment , hVK4-1 segment, hVK5-2 segment, hVK6-21 segment, hVK6D-21 segment, hVK6D-41 segment, hVK7-3 segment, and variants thereof. In some embodiments, a nucleic acid molecule described herein, besides a modified Ig hVκ segment, additional hVκ segments, e.g., hVκ1D-8 segment, hVκ1-9 segment, hVκ1-12 segment, hVκ1D-12 segment, hVκ1 -13 segments, hVκ1D-13 segments, hVκ1-16 segments, hVκ1D-16 segments, hVκ1-17 segments, hVκ1D-17 segments, hVκ1-27 segments, hVκ1-33 segments, hVκ1D-33 segments, hVκ1-37 segments, hVκ1D -37 segments, hVκ1-39 segments, hVκ1D-39, a hVκ1-NL1 segments, hVκ1D-42 segments, hVκ1D-43 segments, hVκ2-4 segments, hVκ2-18 segments, hVκ2D-18 segments, hVκ2-24 segments, hVκ2D -24 segments, hVκ2-28 segments, hVκ2D-28 segments, hVκ2-29 segments, hVκ2D-29 segments, hVκ2-30 segments, hVκ2D-30 segments, hVκ2-40 segments, hVκ2D-40 segments, hVκ2D-26 segments, hVκ3 -7 segments, hVκ3D-7 segments, hVκ3-11 segments, hVκ3D-11 segments, hVκ3-15 segments, hVκ3D-15 segments, hVκ3-20 segments, hVκ3D-20 segments, hVκ4-1 segments, hVκ5-2 segments, hVκ6 -21 segment, and one, more than one, or each of the hVκ6D-21 segment. hVκ1D-8 segment, hVκ1-9 segment, hVκ1-12 segment, hVκ1D-12 segment, hVκ1-13 segment, hVκ1D-13 segment, hVκ1-16 segment, hVκ1D-16 segment, hVκ1-17 segment, hVκ1D-17 segment, hVκ1-27 segment, hVκ1-33 segment, hVκ1D-33 segment, hVκ1-37 segment, hVκ1D-37 segment, hVκ1-39 segment, hVκ1D-39, a hVκ1-NL1 segment, hVκ1D-42 segment, hVκ1D-43 segment, hVκ2-4 segment, hVκ2-18 segment, hVκ2D-18 segment, hVκ2-24 segment, hVκ2D-24 segment, hVκ2-28 segment, hVκ2D-28 segment, hVκ2-29 segment, hVκ2D-29 segment, hVκ2-30 segment, hVκ2D-30 segment, hVκ2-40 segment, hVκ2D-40 segment, hVκ2D-26 segment, hVκ3-7 segment, hVκ3D-7 segment, hVκ3-11 segment, hVκ3D-11 segment, hVκ3-15 segment, hVκ3D-15 segment, In some embodiments comprising more than one or each of the hVK3-20 segment, the hVK3D-20 segment, the hVK4-1 segment, the hVK5-2 segment, the hVK6-21 segment, and the hVK6D-21 segment, the hVK segment is germline is in composition.

In some embodiments, a nucleic acid molecule as described herein (eg, a targeting vector, a non-human animal genome, etc.) may include an Ig light chain κ variable region, eg, in addition to an anchor-modified hVκ segment. , an additional rearranged (non-rearranged) Jκ segment, and in some embodiments, an additional rearranged (non-rearranged) hJκ gene segment. Accordingly, in some embodiments, a recombinant nucleic acid molecule described herein comprises, 5' to 3' in operative linkage: (I) a modified Ig Vκ segment, and (II) one or multiple Ig light chain linkages. Kappa Jκ segments. In some embodiments, a recombinant nucleic acid as described herein (e.g., a targeting vector, a non-human animal genome, etc.) comprises one or more human J _κ segments, e.g., hJκ1, hJκ2, hJκ3, hJκ4, hJκ5, and variants thereof, including one, more than one, or each of these. In some embodiments comprising more than one or each of hJK1, hJK2, hJK3, hJK4, hJK5 segments, the hJK segment is in germline configuration.

Additionally, in some embodiments, a recombinant nucleic acid molecule described herein comprises an operable linker 5' to 3': (I) a modified Ig Vκ segment, and (II) one or multiple Ig light chain linkages. Nucleic acid sequences encoding kappa (Jκ) and Ig light chain constant kappa region (Cκ).

In some embodiments, a germline Ig V segment or variant thereof (eg, encoding FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a modified Ig V segment as described herein) is a germline Ig Light chain variable lambda (Vλ) segments or variants thereof, e.g., human Vλ segments, e.g., hVλ1-36 segments, hVλ1-40 segments, hVλ1-41 segments, hVλ1-44 segments, hVλ1-47 segments, hVλ1- 50 segment, hVλ1-51 segment, hVλ1-62 segment, hVλ2-5 segment, hVλ2-8 segment, hVλ2-11 segment, hVλ2-14 segment, hVλ2-18 segment, hVλ2-23 segment, hVλ2-33 segment, hVλ2- 34 segment, hVλ3-1 segment, hVλ3-9 segment, hVλ3-10 segment, hVλ3-12 segment, hVλ3-13 segment, hVλ3-16 segment, hVλ3-19 segment, hVλ3-21 segment, hVλ3-22 segment, hVλ3- 25 segment, hVλ3-27 segment, hVλ3-31 segment, hVλ3-32 segment, hVλ4-3 segment, hVλ4-60 segment, hVλ4-69 segment, hVλ5-37 segment, hVλ5-39 segment, hVλ5-45 segment, hVλ5- 48 segment, hVλ5-52 segment, hVλ6-57 segment, hVλ7-43 segment, hVλ7-46 segment, hVλ8-61 segment, hVλ9-49 segment, hVλ10-54 segment, hVλ11-55 segment, and variants thereof. In some embodiments, a nucleic acid molecule as described herein is a modified Ig hVλ segment, in addition to an additional hVλ segment, e.g., a hVλ1-36 segment, a hVλ1-40 segment, a hVλ1-41 segment, a hVλ1-44 segment, hVλ1-47 segment, hVλ1-50 segment, hVλ1-51 segment, hVλ1-62 segment, hVλ2-5 segment, hVλ2-8 segment, hVλ2-11 segment, hVλ2-14 segment, hVλ2-18 segment, hVλ2-23 segment, segment hVλ2-33, segment hVλ2-34, segment hVλ3-1, segment hVλ3-9, segment hVλ3-10, segment hVλ3-12, segment hVλ3-13, segment hVλ3-16, segment hVλ3-19, segment hVλ3-21, hVλ3-22 segment, hVλ3-25 segment, hVλ3-27 segment, hVλ3-31 segment, hVλ3-32 segment, hVλ4-3 segment, hVλ4-60 segment, hVλ4-69 segment, hVλ5-37 segment, hVλ5-39 segment, hVλ5-45 segment, hVλ5-48 segment, hVλ5-52 segment, hVλ6-57 segment, hVλ7-43 segment, hVλ7-46 segment, hVλ8-61 segment, hVλ9-49 segment, hVλ10-54 segment, and hVλ11-55 segment Further includes one, more than one, or each of these. hVλ1-36 segment, hVλ1-40 segment, hVλ1-41 segment, hVλ1-44 segment, hVλ1-47 segment, hVλ1-50 segment, hVλ1-51 segment, hVλ1-62 segment, hVλ2-5 segment, hVλ2-8 segment, hVλ2-11 segment, hVλ2-14 segment, hVλ2-18 segment, hVλ2-23 segment, hVλ2-33 segment, hVλ2-34 segment, hVλ3-1 segment, hVλ3-9 segment, hVλ3-10 segment, hVλ3-12 segment, hVλ3-13 segment, hVλ3-16 segment, hVλ3-19 segment, hVλ3-21 segment, hVλ3-22 segment, hVλ3-25 segment, hVλ3-27 segment, hVλ3-31 segment, hVλ3-32 segment, hVλ4-3 segment, hVλ4-60 segment, hVλ4-69 segment, hVλ5-37 segment, hVλ5-39 segment, hVλ5-45 segment, hVλ5-48 segment, hVλ5-52 segment, hVλ6-57 segment, hVλ7-43 segment, hVλ7-46 segment, In some embodiments comprising more than one of, or each of, the hVλ8-61 segment, the hVλ9-49 segment, the hVλ10-54 segment, and the hVλ11-55 segment, the hVλ segment is in germline configuration.

In some embodiments, a nucleic acid molecule as described herein (eg, a targeting vector, a non-human animal genome, etc.) can include an Ig light chain λ variable region, eg, an anchor-modified hVλ segment In addition, additional rearranged (non-rearranged) Jλ segments, and in some embodiments, additional (non-rearranged) hJλ gene segments. Thus, in some embodiments, a recombinant nucleic acid molecule described herein comprises, 5' to 3' in operative linkage: (I) a modified Ig Vλ segment, and (II) one or multiple Ig light chain linkages. Kappa Jλ segment. In some embodiments, a nucleic acid molecule as described herein (e.g., a targeting vector, a non-human animal genome, etc.) comprises one or more human Jλ segments, e.g., hJλ1, hJλ2, hJλ3, hJλ4, hJλ5, hJλ6 , one, more than one, or each of the hJλ7 segment and variants thereof. In some embodiments that include more than one or each of hJλ1, hJλ2, hJλ3, hJλ4, hJλ5, hJλ6, hJλ7 segments, the hJλ segments are in germline configuration. In some embodiments, a recombinant nucleic acid molecule described herein comprises, in operative linkage, 5' to 3': (I) a modified Ig Vλ segment, (II) one or multiple Ig light chain binding lambdas (Jλ). ) segment, and a nucleic acid sequence encoding the Ig light chain constant lambda region (Cλ).

anchor

As described herein, anchors include ligands (or portions thereof) that bind to cognate receptors. In some embodiments, a ligand may be a non-immunoglobulin polypeptide. Thus, as described herein, an anchor may include a non-immunoglobulin polypeptide, eg, a receptor binding portion of a non-immunoglobulin polypeptide. Anchor modifications described herein may be useful for increasing the affinity of an antigen-binding protein, such as an antibody, for a refractory receptor.

Exemplary and well-known non-immunoglobulin polypeptide:cognate receptor pairs include, but are not limited to, non-immunoglobulin polypeptides that bind, for example, to cognate G-protein coupled receptors (GPCRs). Exemplary GPCRs include, but are not limited to, chemokine receptors, glucagon receptors (eg, GLP1:GLP1R), calcitonin receptors, and melanocortin receptors. These and other cognate GPCRs, including non-immunoglobulin polypeptides that bind thereto, are well known in the art. See, for example , Wu et al. (2017) J. Mol. Biol. 429:2726-45, each of which is incorporated herein by reference in its entirety. Additional non-limiting and exemplary non-immunoglobulin polypeptide (ligand):cognate receptor pairs include:

a. Ligands that bind to cognate receptor tyrosine kinases, e.g., ligands such as epidermal growth factor (EGF), insulin, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), etc., but Not limited to this.

b. DLL: Notch Receptor Pair,

c. B7: CD28/CLTA4/PD1 receptor pair,

d. semaphorin:plexin receptor pair,

e. PCSK9/LDLR pair;

f. HLA:LILR pair,

g. HLA:KIR pair,

h. RGD-ligand:integrin pair,

i. Natriuretic peptides (eg, ANP, BNP, CNP, etc.): natriuretic peptide receptor (NPR, NPR3, etc.) pairs, etc.

A ligand:receptor pair may include a protease and an inhibitor.

In some embodiments, the anchor comprises a natriuretic peptide (NP), eg, a receptor binding portion of NP. NP is a peptide of at least eight structurally related amino acids stored as three different prohormones: atrial natriuretic peptide (ANP) prohormone, type B natriuretic peptide (BNP) prohormone, and type C natriuretic peptide (CNP) prohormone. includes Dendroaspis natriuretic peptide, type D natriuretic peptide (DNP), was recently discovered, and its role in humans is still unclear.

ANP prohormone (proANP) is a 126 amino acid polypeptide that is primarily expressed by cardiomyocytes and produces several peptides with blood pressure lowering, natriuretic, diuretic, and/or calliuretic properties. These peptides derived from the ANP prohormone are identified by the amino acid sequence starting at the N-terminal end of the ANP prohormone: e.g., proANPs 1-30 contain persistent NPs, and proANPs 31-67 are vasodilators. and proANPs 79-98 include calliuretic peptides and are identified, in particular , by amino acids 99-126 (also referred to as ANPs). In the kidney, proANP is processed differently, adding four amino acids to the N-terminus, eg proANP 95-126 (also called urodilatin).

BNP prohormone (proBNP) is a 108-amino acid polypeptide that is also expressed primarily by cardiomyocytes. The BNP prohormone is processed in the human heart to form BNP (e.g., amino acids 77-108 of its 108 amino acid prohormone) and NT-proBNP (e.g., amino acids 1-76), both of which are human cycle in BNP is a reliable biomarker of ventricular dilatation. Pandit et al. (2011) Ind.J. Endocrinol. Metab. 15(4) S345-53, incorporated herein by reference in its entirety.

Unlike ANP and BNP, CNP is mainly expressed by endothelial and renal epithelial cells. Two CNP molecules were identified in circulation. In addition, although CNP appears to lack natriuretic function, CNP likely acts as a regulator of vascular tone and growth in either a paracrine or autocrine manner, and there are some indications that CNP may play a role in bone growth. do.

NPs exert their biological functions by specific binding to cell-surface receptors. Three specific receptors have been identified in mammalian tissue: two guanylyl cyclase-coupled receptors (GC-A and GC-B, also referred to as NP receptor (NPR)-A and NPR-B, respectively). NPR-A and NPR-B act through activation of cGMP-dependent signaling cascades. In contrast, the type 3 C receptor (also referred to as NPR-C) does not bind guanylyl cyclase and appears to be primarily involved in the clearance of NP. All three receptors bind ANP, BNP, and CNP with different affinities. The order of ranking of the ligand selectivity for GC-A is ANP ≥ BNP ≫ CNP, the ligand selectivity for GC-B is CNP ≫ ANP ≥ BNP, and the ligand selectivity for NPR-C is ANP > CNP > BNP . Jaubert et al. (1999) PNAS 96(18) 10278-283, incorporated herein by reference in its entirety.

NP receptors may be useful targets for the treatment of hypertension and cardiovascular disease. However, while ANP and BNP have important diuretic, natriuretic and hypotensive properties, CNP may play a role in bone growth. As such, any antibody-based targeting of the NP receptor must take into account the differential binding affinity of the receptor to the NP.

In some embodiments, an anchor comprises the sequence ANP or a portion thereof. The nucleic acid sequence encoding human ANP is presented herein as NCBI Accession No. NM_006172.4 and SEQ ID NO: 1. The amino acid sequence of human ANP is presented herein as NCBI Accession No. NP_006163 and SEQ ID NO: 2. In some embodiments, an anchor described herein comprises a receptor binding portion of ANP, eg, the C-terminal tail of ANP. In some embodiments, the receptor binding portion of ANP comprises the amino acid sequence NSFRY (SEQ ID NO: 3).

linker

In some embodiments, the anchor comprises a linker connecting the receptor binding portion of the non-immunoglobulin polypeptide of interest to FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment, or variants thereof. In some embodiments, a linker can be at least one amino acid in length. In some embodiments, a linker may be two amino acids long. In some embodiments, a linker can be 3 amino acids long. In some embodiments, the linker can be 4 amino acids long, for example, the linker can include the sequence GLSG (SEQ ID NO: 13). In some embodiments, the linker can be 5 amino acids long, for example, the linker can include the sequence GGGGS (SEQ ID NO: 5). In some embodiments, the linker can be 6 amino acids long, for example the linker can include the sequence GLSGSG (SEQ ID NO: 14). In some embodiments, a linker may be 7 amino acids long. In some embodiments, a linker may include the sequence GLSGLSGS (SEQ ID NO: 15). In some embodiments, a linker may be 9 amino acids long. In some embodiments, the linker can be 10 amino acids long, for example, the linker can include GLSGLSGLSG (SEQ ID NO: 16) or GLSGGSGLSG (SEQ ID NO: 17). In some embodiments, the first and second linkers are the same length and are each greater than 10 amino acids in length.

In some embodiments, a recombinant nucleic acid molecule described herein comprises a sequence set forth as SEQ ID NO: 8 or a degenerate variant thereof, or SEQ ID NO: 10 or a degenerate variant thereof.

targeting vector

Further provided are targeting vectors for use in the methods of making genetically modified non-human animals, cells, tissues or embryos provided herein.

In one embodiment, an insert nucleic acid, e.g., as described herein, flanked by 5' and 3' homologous arms capable of undergoing homologous recombination with a locus of interest, e.g., an Ig heavy or light chain variable region locus. A targeting vector comprising a recombinant nucleic acid molecule comprising a modified Ig V segment as described above is provided. Examples of targeting vectors and components of targeting vectors (ie, insert nucleic acids, polynucleotides of interest, expression cassettes, etc.) are described in detail herein below.

Homologous arms and target sites (i.e., cognate genomic regions) are "complementary" or "complementary" to each other when the two regions share a sufficient level of sequence identity to each other to act as substrates for homologous recombination reactions. It is "complementary". The term “homology” refers to DNA sequences that are identical or share sequence identity with a corresponding or “complementary” sequence. Sequence identity between a given target site and the corresponding homology arms found in the targeting vector can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity shared by the homology arms of the targeting vector (or fragments thereof) and the target site (or fragments thereof) is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Additionally, the complementary region of homology between the homology arm and the complementary target site may be of any length sufficient to promote homologous recombination at the truncated recognition site. For example, a given homology arm and/or complementary target region is at least 5-10 kb, 5-15 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70 kb. to 80 kb, 80 to 90 kb, 90 to 100 kb, 100 to 110 kb, 110 to 120 kb, 120 to 130 kb, 130 to 140 kb, 140 to 150 kb, 150 to 160 kb, 160 to 170 kb, 170 to 180 k b, 180 to 190 kb, 190 to 200 kb , a complementary region of homology that is between 200 kb and 300 kb or more in length (eg, as described in vectors described elsewhere herein) such that the homology arm is capable of homologous recombination with the corresponding target site in the cell genome. has sufficient homology to perform For ease of reference, homology arms are referred to herein as 5' and 3' homology arms. This term relates to the position of the homology arms relative to the insert nucleic acid within the targeting vector.

Thus, the homology arms of the targeting vector are designed to be complementary to the target site with the targeted locus. Thus, homology arms may be complementary to loci that are natural for the cell, or alternatively they may be complementary to regions of heterologous or exogenous segments of DNA integrated into the genome of the cell, which may include transgenes, expression of genomic DNA cassettes, or heterologous or exogenous regions, but are not limited thereto. Alternatively, the homology arm of the targeting vector may be complementary to a region of a human artificial chromosome or any other engineered genomic region contained in a suitable host cell. In addition, the homology arms of the targeting vector may correspond to or be derived from regions of a BAC library, a cosmid library, or a P1 phage library. Thus, in certain embodiments, a homology arm of a targeting vector is complementary to a eukaryotic, non-human, mammalian, non-human mammalian, human, rodent, mouse or rat genomic locus that is native, heterologous or exogenous to a given cell. . In some embodiments, homology arms are derived from synthetic DNA.

In some targeting vector embodiments, the targeting vector is such that, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus converts the non-human Ig C _H at the non-human Ig heavy chain locus. 5 targeting a non-human Ig heavy chain locus to include a recombinant nucleic acid molecule (a modified Ig V _H segment, and optionally a recombinant nucleic acid molecule comprising an Ig D _H and/or Ig J _H segment) with an operative linkage upstream. ' and 3' homology arms, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or wherein the non-human Ig heavy chain locus is a human or humanized immunoglobulin heavy chain variable region, an endogenous deletion of the Ig V _H , D _H , and/or J _H gene segment, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule substitutes a non-human V _H segment at the non-human Ig heavy chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule comprises one or more non-human V _H segments, all non-human D H segments, and all non-human D _H segments at the non-human Ig heavy chain locus. Substitute all non-human J _H segments. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule _is one non-human V H segment, all non-human V _H segments, all non-human Ig heavy chain loci at the non-human Ig heavy chain locus. Substitute all but non-human D _H segments, and all non-human J _H segments. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus recombines operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus. Contains nucleic acid molecules.

In some embodiments, a targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and a non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus is non-human Ig heavy chain locus. - a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid comprising a modified Ig V _H segment, and optionally an Ig D _H segment, an Ig J _H segment and/or an Ig C _H gene) operably linked to a human Ig heavy chain regulatory sequence. molecule), optionally wherein the non-human Ig heavy chain locus is a rodent or rodent cell (eg, a rodent embryonic stem cell) of an endogenous rodent Ig heavy chain locus and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, a deletion of endogenous Ig V _H , D _H , and/or J _H gene segments, or a combination thereof. and, optionally, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule comprises at least one non-human V _H segment at the non-human Ig heavy chain locus, all non-human D _H gene segments, All non-human J _H gene segments, and at least one non-human C _H gene are replaced.

In some embodiments, the 5' homology arm comprises the sequence presented as SEQ ID NO: 12 and/or the 3' homology arm comprises the sequence presented as SEQ ID NO: 13.

In some targeting vector embodiments, the targeting vector may, upon homologous recombination between a recombinant nucleic acid molecule described herein and the targeting vector and a non-human Ig light chain locus, cause the targeted non-human Ig light chain locus to replace the non-human Ig light chain locus. to include a recombinant nucleic acid molecule (eg, a recombinant nucleic acid molecule comprising a modified Ig V _L segment, and optionally an Ig J _L segment described herein) in an operable linkage upstream of a non-human Ig C _L in optionally comprising 5' and 3' homology arms targeting a non-human Ig light chain locus, wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or the non-human Ig light chain locus is a human or humanized deletions of immunoglobulin light chain variable regions, endogenous Ig V _L and/or J _L gene segments, or combinations thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces the non-human V _L segment at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule substitutes one or more non-human V _L segments and all non-human J _L segments at the non-human Ig light chain locus. do. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule displaces all non-human V _L segments and all non-human J _H segments at the non-human Ig light chain locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus produces a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus. include

In some embodiments, a targeting vector described herein is a nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and a non-human Ig light chain locus, the targeted non-human Ig light chain locus is a non-human Ig light chain locus. A recombinant nucleic acid molecule (e.g., comprising a modified Ig V _L segment, and optionally an Ig J _L segment and/or Ig C _L gene as described herein) operably linked at the locus to a non-human Ig light chain regulatory sequence. 5' and 3' homology arms targeting a non-human Ig light chain locus to include a recombinant nucleic acid molecule), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or is a non-human Ig light chain locus. The light chain locus comprises a human or humanized immunoglobulin light chain variable region, deletion of endogenous Ig V _L and/or J _L gene segments, or a combination thereof, optionally homology between the targeting vector and the non-human Ig light chain locus. Upon recombination, the recombinant nucleic acid molecule replaces the non-human V _L segment, all non-human J _L gene segments, and the non-human C _L gene at the non-human Ig light chain locus.

In some targeting vector embodiments, the targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus becomes the non-human Ig light chain κ locus. A recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig VK segment, and optionally an Ig JK segment as described herein) with an operable linkage upstream of a non-human Ig CK at the light chain κ locus. 5' and 3' homology arms targeting a non-human Ig light chain κ locus to include, optionally wherein the non-human Ig light chain κ locus is an endogenous rodent Ig light chain κ locus, and/or the non-human Ig light chain The κ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig Vκ and/or Jκ gene segments, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule replaces the non-human Vκ segment at the non-human Ig light chain κ locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule displaces one or more non-human Vκ segments and all non-human Jκ segments at the non-human Ig light chain κ locus. do. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule displaces all non-human Vκ segments and all non-human Jκ segments at the non-human Ig light chain κ locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus is operably linked to a non-human Ig light chain κ regulatory sequence at the Ig light chain κ locus. Includes recombinant nucleic acid molecules.

In some targeting vector embodiments, the targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus is the Ig light chain κ locus. A recombinant nucleic acid molecule (e.g., a modified Ig Vκ segment, and optionally a recombinant Ig Jκ segment and/or Ig Cκ gene as described herein) operably linked to a non-human Ig light chain κ regulatory sequence in nucleic acid molecule), optionally wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid The molecule replaces the non-human VK segment at the non-human Ig light chain κ locus, all non-human JK gene segments, and the non-human CK gene.

In some targeting vector embodiments, the targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus becomes the non-human Ig Non-human Ig light chain to include a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig Vλ segment, and optionally an Ig Jλ segment) with an operable linkage upstream of the non-human Ig Cλ at the light chain locus. optionally wherein the non-human Ig light chain λ locus is an endogenous rodent Ig light chain λ locus and/or the non-human Ig light chain λ locus is a human or humanized deletion of an immunoglobulin light chain variable region, endogenous Ig Vλ and/or Jλ gene segments, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule replaces the non-human Vλ segment at the non-human Ig light chain λ locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule replaces one or more non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule displaces all non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain λ locus. . In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus is operably linked to a non-human Ig light chain λ regulatory sequence at the Ig light chain λ locus. Includes recombinant nucleic acid molecules.

In some embodiments, the targeting vector is a recombinant nucleic acid molecule described herein, and upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus is displaced from the Ig light chain λ locus. - a recombinant nucleic acid molecule comprising a modified Ig Vλ segment operably linked to a human Ig light chain λ regulatory sequence, and optionally an Ig Jλ segment and/or Ig Cλ gene as described herein ) and 5' and 3' homology arms targeting the non-human Ig light chain λ locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule is a non-human Vλ segment at the non-human Ig light chain λ locus, all non-human Jλ gene segments, and a non-human Jλ gene segment at the non-human Ig light chain λ locus. -Substitute the human Cλ gene.

In some embodiments, a targeting vector as described herein may further comprise a nucleotide sequence flanked by a site-specific recombination targeting sequence. It is recognized that any region or individual polynucleotide of interest within the targeting vector may also be flanked by such sites. A site-specific recombinase can be introduced into a cell by any means, including introducing a recombinase polypeptide into the cell or introducing a polynucleotide encoding the site-specific recombinase into a host cell. . A polynucleotide encoding a site-specific recombinase can be located within a targeting vector or within a separate polynucleotide. A site-specific recombinase is a cell, including, for example, an inducible promoter, a promoter endogenous to a cell, a promoter heterologous to a cell, a cell-specific promoter, a tissue-specific promoter, or a developmental stage-specific promoter. can be operably linked to a promoter active in Site-specific recombination target sequences that can be flanked by nucleotide sequences or any polynucleotide of interest in the targeting vector include loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att , FRT, ro x, or combinations thereof, but is not limited thereto.

In some embodiments, a site-specific recombination site is flanked by a polynucleotide encoding a selectable marker in a targeting vector. In this case, after integration of the targeting vector at the targeted locus, the nucleotide sequence between the site-specific recombination site can be removed.

In some embodiments, targeting vectors as described herein include selectable markers that may be contained in a selectable cassette. These selectable markers include neomycin phosphotransferase (neo ^r ), hygromycin B phosphotransferase (hyg ^r ), puromycin-N-acetyltransferase (puro ^r ), blastidin S diamina (bsr ^r ), xanthine/guanine phosphoribosyl transferase (gpt), or herpes simplex virus thymidine kinase (HSV-k). In one embodiment, a polynucleotide encoding a selectable marker may be operably linked to a promoter active in the cell. In one embodiment, a polynucleotide encoding a selectable marker is flanked by a site-specific recombination target sequence.

non-human animal genome

Also described herein are non-human animal genomes comprising recombinant nucleic acid molecules and/or targeting vectors as described herein. In some non-human animal genome examples, the non-human animal genome comprises a recombinant nucleic acid molecule as described herein at an endogenous Ig locus of the non-human animal genome, e.g., the non-human animal genome , wherein the targeting vector comprises 5' and 3' homology arms that target an endogenous Ig locus. In some embodiments, the non-human animal genome is a rodent genome. In some embodiments, the non-human animal genome is a rat genome. In some embodiments, the non-human animal genome is a mouse genome.

In a non-human animal genome example, the genome comprises a recombinant nucleic acid molecule ( _{e.g., a modified Ig V H segment} , and, optionally, a recombinant nucleic acid molecule comprising an Ig D _H and/or an Ig J _H segment), optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus, and/or is a non-human Ig heavy chain locus. - The human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, a deletion of endogenous Ig V _H , D _H , and/or J _H gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human V _H segment at a non-human Ig heavy chain locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human V _H segments, all non-human D _H segments, and all non-human J _H segments at a non-human Ig heavy chain locus. In some embodiments, a recombinant nucleic acid molecule comprises one or all non-human V _H segments, all non-human D _H segments, and all non-human J _H segments at a non-human Ig heavy chain locus _. Replace everything except In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig heavy chain regulatory sequence at a non-human Ig heavy chain locus.

In some embodiments, the non-human Ig heavy chain locus is operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus in a recombinant nucleic acid molecule (e.g., a modified Ig V _H segment, and optionally an Ig a recombinant nucleic acid molecule comprising a D _H segment, an Ig J _H segment and/or an Ig C _H gene), optionally wherein the non-human Ig heavy chain locus is a rodent or rodent cell (eg, a rodent embryonic stem cell) The endogenous rodent Ig heavy chain locus at and/or the non-human Ig heavy chain locus is a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V _H , deletion of D _H , and/or J _H gene segments, or combinations thereof. and optionally, the recombinant nucleic acid molecule comprises one or more non-human V _H segments, all non-human D _H gene segments, all non-human J _H gene segments, and one or more non-human C gene segments at a non-human Ig heavy chain locus. Replace _{the H} gene.

In a non-human animal genome example, the genome comprises a recombinant nucleic acid molecule (e.g., a modified Ig V _L segment, and optionally a modified Ig V _L segment, and optionally a a recombinant nucleic acid molecule comprising an Ig J _L segment as described in ), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or is a non-human The Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of the endogenous Ig V _L and/or J _L gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human V _L segment at a non-human Ig light chain locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human V _L segments and all non-human J _L segments at the non-human Ig light chain locus. In some embodiments, the recombinant nucleic acid molecule substitutes all non-human V _L segments and all non-human J _H segments at the non-human Ig light chain locus. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain regulatory sequence at an Ig light chain locus.

In non-human animal genomic examples, the Ig light chain locus is operably linked to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus by a recombinant nucleic acid molecule (e.g., a modified Ig V _L segment, and optionally a recombinant nucleic acid molecule comprising an Ig J _L segment and/or an Ig C _L gene as described herein), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or a non-human Ig light chain locus. The light chain locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig V _L and/or J _L gene segments, or a combination thereof, optionally wherein the recombinant nucleic acid molecule is non-human Ig light chain locus. - replaces the human V _L segment, all non-human J _L gene segments, and the non-human _CL gene.

In a non-human animal genome example, the genome is composed of a recombinant nucleic acid molecule (e.g., a modified Ig Vκ segment, and optionally a modified Ig Vκ segment as described herein) with an operable linkage upstream of the non-human Ig Cκ at the non-human Ig light chain κ locus. a recombinant nucleic acid molecule comprising an Ig Jκ segment as described), optionally wherein the non-human Ig light chain κ locus is an endogenous rodent Ig light chain κ locus, and/or a non-human Ig light chain κ locus. The human Ig light chain κ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig Vκ and/or Jκ gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human Vκ segment at a non-human Ig light chain κ locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human VK segments and all non-human JK segments at the non-human Ig light chain κ locus. In some embodiments, the recombinant nucleic acid molecule substitutes all non-human VK segments and all non-human JK segments at the non-human Ig light chain κ locus. In some embodiments, a recombinant nucleic acid molecule operably linked to a non-human Ig light chain κ regulatory sequence at an Ig light chain κ locus.

In some embodiments, a recombinant nucleic acid molecule (e.g., a modified Ig VK segment, and optionally an Ig JK segment as described herein and / or a recombinant nucleic acid molecule comprising an Ig CK gene, optionally wherein the recombinant nucleic acid molecule comprises a non-human VK segment, all non-human JK gene segments, and a non-human CK gene at the non-human Ig light chain κ locus. Substitute

In a non-human animal genome example, the genome comprises a recombinant nucleic acid molecule (e.g., a modified Ig Vλ segment, and optionally an Ig Jλ segment) with an operative linkage upstream of the non-human Ig Cλ at the non-human Ig light chain locus. a recombinant nucleic acid molecule comprising a), optionally wherein the non-human Ig light chain λ locus is an endogenous rodent Ig light chain λ locus and/or the non-human Ig light chain λ locus is a human or deletion of humanized immunoglobulin light chain variable regions, endogenous Ig Vλ and/or Jλ gene segments, or combinations thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human Vλ segment at a non-human Ig light chain λ locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule displaces all non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain λ locus. . In some embodiments, a recombinant nucleic acid molecule operably linked to a non-human Ig light chain λ regulatory sequence at an Ig light chain λ locus.

In some non-human animal genomic examples, the non-human Ig light chain λ locus is a recombinant nucleic acid molecule (e.g., a modified Ig Vλ segment, and / or a recombinant nucleic acid molecule comprising an Ig Jλ segment and / or an Ig Cλ gene as described herein). In some embodiments, the recombinant nucleic acid molecule replaces a non-human Vλ segment at a non-human Ig light chain λ locus, all non-human Jλ gene segments, and a non-human Cλ gene.

Non-human animal cells, non-human animals, and methods of making the same

Non-human animals and non-human animal cells

Non-human animals and non-human animal cells that express anchor-modified immunoglobulins are provided, eg, comprising an Ig leader sequence and the framework (FR) and complementarity determining regions (CDRs) of germline V segments. ) a modified Ig V segment, e.g., an Ig heavy chain variable region (V _H ) segment or an Ig light chain variable region (V _L ) segment, modified to encode an anchor in an operative linkage therewith between sequences. It is derived from a locus modified to contain a recombinant nucleic acid molecule as described. Thus, non-human animals, embryos, cells comprising recombinant nucleic acid molecules, targeting constructs, and/or animal genomes described herein are provided.

In some non-human animal or non-human animal cell embodiments, the non-human animal or cell comprises a recombinant nucleic acid molecule as described herein (Ig leader sequence and the frame of a germline V segment located randomly within the animal's genome). A modified Ig V segment, e.g., an Ig heavy chain variable region (V _H ) segment or an Ig light chain variable region ( V _L ) segment). In some embodiments, a non-human animal is a recombinant nucleic acid molecule as described herein (Ig leader sequence, framework (FR) and complementarity determining regions (CDRs) of germline V segments at the endogenous Ig locus of the non-human animal. ) a modified Ig V segment, eg, comprising an Ig heavy chain variable region (V _H ) segment or an Ig light chain variable region (V _L ) segment, modified to encode an anchor with an operative linkage between sequences) and, for example, the non-human animal comprises a targeting vector as described herein, wherein the targeting vector comprises 5' and 3' homology arms that target an endogenous Ig locus. In some embodiments, the non-human animal is a rodent. In some embodiments, non-human animal cells are rodent cells. In some embodiments, the non-human animal is a rat. In some embodiments, non-human animal cells are rat cells. In some embodiments, a non-human animal is a mouse. In some embodiments, non-human animal cells are mouse cells.

In an embodiment, the non-human animal or non-human animal cell is a recombinant nucleic acid molecule (e.g., a modified Ig V _H segment, a modified Ig V _H segment, and optionally a recombinant nucleic acid molecule comprising an Ig D _H and/or Ig J _H segment), optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or or the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, a deletion of endogenous Ig V _H , D _H , and/or J _H gene segments, or combinations thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human V _H segment at a non-human Ig heavy chain locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human V _H segments, all non-human D _H segments, and all non-human J _H segments at a non-human Ig heavy chain locus. In some embodiments, a recombinant nucleic acid molecule comprises one or all non-human V _H segments, all non-human D _H segments, and all non-human J _H segments at a non-human Ig heavy chain locus _. Replace everything except In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig heavy chain regulatory sequence at a non-human Ig heavy chain locus.

In some embodiments, the non-human Ig heavy chain locus is operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus in a recombinant nucleic acid molecule (e.g., a modified Ig V _H segment, and optionally an Ig a recombinant nucleic acid molecule comprising a D _H segment, an Ig J _H segment and/or an Ig C _H gene), optionally wherein the non-human Ig heavy chain locus is a rodent or rodent cell (eg, a rodent embryonic stem cell) The endogenous rodent Ig heavy chain locus at and/or the non-human Ig heavy chain locus is a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V _H , deletion of D _H , and/or J _H gene segments, or combinations thereof. and optionally wherein the recombinant nucleic acid molecule comprises one or more non-human V _H segments, all non-human D _H gene segments, all non-human J _{H gene segments, and one or more non-human V H} gene segments at a non-human Ig heavy chain locus. Replace the C _H gene.

In some embodiments, the non-human animal or non-human animal cell is a recombinant nucleic acid molecule (e.g., a modified Ig V _L segment) with an operative linkage upstream of the non-human Ig C _L at the non-human Ig light chain locus. , and optionally a recombinant nucleic acid molecule comprising an Ig J _L segment as described herein), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and Alternatively, the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig V _L and/or J _L gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human V _L segment at a non-human Ig light chain locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human V _L segments and all non-human J _L segments at the non-human Ig light chain locus. In some embodiments, the recombinant nucleic acid molecule substitutes all non-human V _L segments and all non-human J _H segments at the non-human Ig light chain locus. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain regulatory sequence at an Ig light chain locus.

In some embodiments, the Ig light chain locus is operatively linked to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus in a recombinant nucleic acid molecule (e.g., a modified Ig V L segment, and, optionally, a modified Ig V _L segment as described herein). a recombinant nucleic acid molecule comprising an Ig J _L segment and/or an Ig C _L gene as described above, optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or the non-human Ig light chain locus is A human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig V _L and/or J _L gene segments, or a combination thereof, optionally wherein the recombinant nucleic acid molecule is at a non-human Ig light chain locus at a non-human V L locus. _{The L} segment, all non-human J _L gene segments, and the non-human C _L gene are replaced.

In some embodiments, the non-human animal or non-human animal cell is a recombinant nucleic acid molecule (e.g., a modified Ig Vκ segment, a modified Ig Vκ segment, and optionally a recombinant nucleic acid molecule comprising an Ig Jκ segment as described herein), optionally wherein the non-human Ig light chain κ locus is an endogenous rodent Ig light chain κ locus. and/or the non-human Ig light chain κ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig Vκ and/or Jκ gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human Vκ segment at a non-human Ig light chain κ locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human VK segments and all non-human JK segments at the non-human Ig light chain κ locus. In some embodiments, the recombinant nucleic acid molecule substitutes all non-human VK segments and all non-human JK segments at the non-human Ig light chain κ locus. In some embodiments, a recombinant nucleic acid molecule operably linked to a non-human Ig light chain κ regulatory sequence at an Ig light chain κ locus.

In some embodiments, a recombinant nucleic acid molecule (comprising a modified Ig Vκ segment, and optionally an Ig Jκ segment(s) and/or an Ig CK gene as described herein) is non-specific at the Ig light chain κ locus. -operably linked to a human Ig light chain κ regulatory sequence, optionally wherein the recombinant nucleic acid molecule comprises a non-human Vκ segment at the non-human Ig light chain κ locus, all non-human Jκ gene segments, and a non-human CK gene Replace

In some embodiments, the non-human animal or non-human animal cell is a recombinant nucleic acid molecule (e.g., a modified Ig Vλ segment, and optionally comprising a non-human Ig light chain λ locus comprising a recombinant nucleic acid molecule comprising an Ig Jλ segment), optionally wherein the non-human Ig light chain λ locus is an endogenous rodent Ig light chain λ locus and/or is a non-human Ig The light chain λ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of an endogenous Ig Vλ and/or Jλ gene segment, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule substitutes a non-human Vλ segment at a non-human Ig light chain λ locus. In some embodiments, the recombinant nucleic acid molecule substitutes one or more non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule displaces all non-human Vλ segments and all non-human Jλ segments at the non-human Ig light chain λ locus. . In some embodiments, a recombinant nucleic acid molecule operably linked to a non-human Ig light chain λ regulatory sequence at an Ig light chain λ locus.

In some embodiments, the non-human Ig light chain λ locus is operatively linked to a non-human Ig light chain λ regulatory sequence at the Ig light chain λ locus in a recombinant nucleic acid molecule (e.g., a modified Ig Vλ segment, and optionally a modified Ig Vλ segment herein). Ig Jλ segment and/or Ig Cλ gene as described). In some embodiments, the recombinant nucleic acid molecule replaces a non-human Vλ segment at a non-human Ig light chain λ locus, all non-human Jλ gene segments, and a non-human Cλ gene.

Methods of Making Non-Human Animals or Non-Human Animal Cells

Also described are methods of using a recombinant nucleic acid molecule, eg, a targeting vector, as described herein to produce non-human cells, non-human embryos, and/or non-human animals in vitro . In some embodiments, an in vitro method of transforming an isolated cell involves introducing a recombinant nucleic acid molecule as described herein into the isolated cell, eg, by contacting the cell with a targeting vector as described herein. Include steps. In some embodiments, a cell is a host cell. In some embodiments, the cell is an embryonic stem (ES) cell. In some embodiments, a cell as described herein or a cell made according to a method described herein is a rodent cell, eg, the rodent cell is a rat cell or a mouse cell.

Also described are methods of using nucleic acid molecules, non-human cells, and/or non-human animals as described herein to make anchor-modified antigen-binding proteins. Also described are non-human animal embryos and non-human animals that may comprise and/or may develop (eg, generate) from embryonic stem cells as described herein. Such embryos or non-human animals can be prepared by transplanting an ES cell as described herein into an embryo and/or transplanting an embryo comprising an ES cell into a suitable host and developing the ES cell or embryo into a viable progeny. It can be developed by a method comprising the step of maintaining the host under suitable conditions for a period of time.

As described herein, targeting vectors can be used to target Ig loci having human or humanized immunoglobulin variable regions. Immunoglobulin loci comprising human variable region gene segments are known in the art and are described in, for example, U.S. Patent Nos. 5,633,425; 5,770,429; 5,814,318; 6,075,181; 6,114,598; 6,150,584; 6,998,514; 7,795,494; 7,910,798; 8,232,449; 8,502,018; 8,697,940; 8,703,485; 8,754,287; 8,791,323; 8,809,051; 8,907,157; 9,035,128; 9,145,588; 9,206,263; 9,447,177; 9,551,124; 9,580,491 and 9,475,559, each of which is incorporated herein by reference in its entirety, as well as U.S. Patent Publication Nos. No. 287 and No. 2015/0113668, each of which is incorporated herein by reference in its entirety, and PCT Publication Nos. WO2007117410, WO2008151081, WO2009157771, WO2010039900, WO2011004192, WO2011123708 and WO2014093908, each of which is incorporated herein by reference in its entirety). there is.

In some embodiments, a non-human animal as described herein comprises an exogenous fully human immunoglobulin transgene comprising a modified Ig V segment as described herein, which can be rearranged in a mouse precursor B cell. (Alt et al., 1985, Immunoglobulin genes in transgenic mice, Trends Genet 1:231-236; incorporated herein by reference in its entirety). In these examples, a fully human immunoglobulin transgene comprising a modified Ig V segment as described herein can be inserted (randomly), and the endogenous immunoglobulin gene can also be knocked out (Green et al., 1994 , Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs, Nat Genet 7:13-21;Lonberg et al., 1994, Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 368:856- 859; Jakobovits et al., 2007, From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice, Nat Biotechnol 25:1134-1143; each of these publications is incorporated herein by reference in its entirety), e.g., herein Endogenous immunoglobulin heavy chain and κ light chain loci are inactivated, for example, by targeted deletion of small but important portions of each endogenous locus followed by introduction of human immunoglobulin loci as randomly integrated large transgenes or small chromosomes (Tomizuka et al. , 2000, Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies, PNAS USA 97:722-727; incorporated by reference in its entirety).

In some embodiments, human or humanized immunoglobulin heavy and light chain loci comprising modified Ig V segments as described herein are located at endogenous immunoglobulin heavy and light chain loci, respectively. It has also been shown that even substitution of a single endogenous _VH gene segment with a human _VH gene segment can generate an immune-response comprising a humanized immunoglobulin variable domain. See, eg , Tien et al. (2016) Cell 166:1471-84; the entirety of which is incorporated herein by reference.

Methods have previously been described for large- scale in situ gene replacement of mouse germline immunoglobulin variable loci with human germline immunoglobulin variable loci while maintaining the mouse's ability to produce offspring. See, eg, US Pat. Nos. 6,596,541 and 8,697,940, each of which is incorporated herein by reference in its entirety. Specifically, precise substitutions of 6 megabases of both the mouse heavy and κ light chain immunoglobulin variable loci with their human counterparts are described while leaving the mouse constant regions intact. As a result, a mouse was generated in which the entire germline immunoglobulin variable repertoire of the mouse was correctly substituted with an equivalent human germline immunoglobulin variable sequence while maintaining the mouse constant region. Human variable regions are linked to mouse constant regions to form chimeric human-mouse immunoglobulin loci that are rearranged and expressed at physiologically relevant levels. The expressed antibody is “reverse chimeric”, ie, contains a human variable region sequence and a mouse constant region sequence.

In some embodiments, a mouse having a humanized immunoglobulin variable region and a mouse constant region expressing an antibody having a human or humanized variable region is referred to as a VELOCIMMUNE® mouse. VELOCIMMUNE® humanized mice display a fully functional humoral immune system that is essentially indistinguishable from wild-type mice. They represent normal cell populations at all stages of B cell development. They represent normal lymphoid organ morphology. Antibody sequences from VELOCIMMUNE® mice show normal V(D)J rearrangements and normal somatic hypermutation frequencies. The antibody populations of these mice reflect an isotype distribution due to normal class switching (eg, normal isotype cis-switching). Immunization of VELOCIMMUNE® mice results in a robust humoral immune-response that generates a large and diverse repertoire of antibodies with human immunoglobulin variable domains suitable for use as candidate therapeutics. This platform provides a rich source of naturally affinity-matured human immunoglobulin variable region sequences for the production of pharmaceutically acceptable antibodies and other antigen-binding proteins. This is precisely the substitution of mouse immunoglobulin variable sequences with human immunoglobulin variable sequences such that endogenous non-human constant region gene sequences and human immunoglobulin variable sequences are operably linked in an inverse chimera fashion that allows for the production of VELOCIMMUNE® mice. is to do

Mice that have been modified in a reverse chimeric fashion have a humanized variable region (e.g., (D), J and one or more human V gene segments operably linked to, but not limited to, an endogenous constant region at an endogenous immunoglobulin locus). Including a mouse modified to include), for example,

(a) at the endogenous heavy chain locus:

(i) an unrearranged human (localized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the unrearranged human (localized) immunoglobulin heavy chain variable region comprises a plurality of unrearranged human heavy chain A variable region _VH gene segment (e.g., an unrearranged human _VH gene segment that is a fully functional human), one or more unrearranged immunoglobulin heavy chain _DH gene segments, and one or more unrearranged immunoglobulin heavy chain J contains _{an H} gene segment;

Optionally, the one or more unrearranged immunoglobulin heavy chain D _H gene segments and the one or more unrearranged immunoglobulin heavy chain J _H gene segments are selected from one or more unrearranged human immunoglobulin heavy chain D _H gene segments (e.g., is a fully functional human D _H gene segment) and/or is one or more unrearranged human immunoglobulin heavy chain J _H gene segments (eg, is a fully functional human J _H gene segment);

(ii) a restricted rearranged human (localized) heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the restricted unrearranged human (localized) heavy chain variable region comprises one or more unrearranged immunoglobulin D _H genes. segment and one unrearranged human heavy chain variable region V _H gene segment operably linked with one or more unrearranged immunoglobulin heavy chain J _H gene segments; optionally, one or more unrearranged immunoglobulin heavy chain segments. The D _H gene segment and one or more unrearranged immunoglobulin heavy chain J _H gene segments are each one or more unrearranged human immunoglobulin heavy chain D _H gene segments and/or one or more unrearranged human immunoglobulin heavy chain J _H gene segments. segment);

(iii) a histidine modified unrearranged human (localized) heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the histidine modified human (localized) heavy chain variable region comprises a histidine codon and at least one non-histidine codon. an unrearranged immunoglobulin heavy chain variable gene sequence comprising a substitution of or insertion of at least one histidine codon into a complementarity determining region 3 (CDR3) coding sequence); or

(iv) a heavy chain-only immunoglobulin coding sequence comprising an unrearranged human (localized) heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the endogenous heavy chain constant region is (1) an IgM isoform associated with a light chain. and (2) a non-IgM gene lacking a sequence encoding a functional CH1 domain, e.g., an IgG gene; wherein the non-IgM gene is capable of being covalently linked with a light chain constant region. encodes a non-IgM isoform lacking the CH1 domain);

and/or

(b) at the endogenous light chain locus:

(i) an unrearranged human (localized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region, wherein the unrearranged human (localized) immunoglobulin light chain variable region comprises a plurality of unrearranged human light chain a variable region V _L gene segment (e.g., an unrearranged human V _L gene segment that is a fully functional human) and one or more unrearranged immunoglobulin light chain J _L gene segments;

Optionally, the one or more unrearranged immunoglobulin light chain J _L gene segments are one or more unrearranged human immunoglobulin light chain J _L gene segments (eg, fully functional human J _H L gene segments);

Optionally, the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain kappa (κ) locus and the unrearranged human (localized) immunoglobulin light chain variable region is a human variable κ (Vκ) and binding κ (Jκ) gene segment; The endogenous light chain constant region is an endogenous κ chain constant region sequence; the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain lambda (λ), the unrearranged human (localized) immunoglobulin light chain variable region comprises human variable λ (V _λ ) and binding λ (J _λ ) gene segments; endogenous light chain constant region is an endogenous λ chain constant region sequence; Optionally, the endogenous immunoglobulin light chain λ locus comprises (a) one or more human V _λ gene segments, (b) one or more human J _λ gene segments, and (c) one or more human C _λ gene segments, wherein (a) and (b) are (c) and rodent immunoglobulin light chain constant (C _λ ) is operably linked to a gene segment; the endogenous immunoglobulin λ light chain locus comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ), and one or more human immunoglobulin λ light chain enhancers (Eλ), optionally comprising three human Eλ;

(ii) a consensus light chain coding sequence comprising a rearranged human (localized) light chain variable region sequence operably linked to an endogenous light chain constant region, wherein the rearranged human (localized) light chain variable region sequence is an immunoglobulin light chain J _L gene segment and rearranged human light chain variable region V _L );

(iii) a restricted rearranged human (localized) light chain variable region operably linked to an endogenous light chain constant region, wherein the restricted rearranged human (localized) light chain variable region binds to one or more rearranged human immunoglobulin light chain (J _L ) comprising no more than two unrearranged human immunoglobulin light chain variable (V _L ) gene segments operably linked to the gene segment;

(iv) a histidine modified rearranged human (localized) light chain variable region operably linked to an endogenous light chain constant region, wherein the histidine modified unrearranged human (localized) light chain variable region has a histidine codon and at least one an unrearranged human (localized) immunoglobulin light chain variable region sequence comprising a substitution of a non-histidine codon or an insertion of at least one histidine codon into a complementarity determining region 3 (CDR3) coding sequence); or

(v) a histidine modified rearranged human (localized) light chain variable region operably linked to an endogenous light chain constant region, wherein the histidine modified rearranged human (lower) light chain variable region has at least one non-histidine codon. A mouse comprising a rearranged human (localized) immunoglobulin light chain variable region sequence comprising a substitution of a histidine codon or an insertion of at least one histidine codon into a complementarity determining region 3 (CDR3) coding sequence,

Optionally, the mouse

(i) a human (localized) immunoglobulin heavy chain locus comprising a functional ADAM6 locus such that the mouse exhibits wild-type fertility of a non-human animal; and/or

(ii) optionally further comprising an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity such that at least 10% of the rearranged variable region genes comprise non-template additions. mouse,

has been described previously. See, for example , US Patent No. 8,697,940; 8,754,287; 9,204,624; 9,334,334; 9,801,362; 9,332,742; and 9,516,868; US Patent Publication Nos. 20110195454, 20120021409, 20120192300, 20130045492; 20150289489; 20180125043; 20180244804; PCT Publication Nos. WO2019/113065, WO2017210586 and WO2011163314; See Lee et al. (2014) Nature Biotechnology 32:356, each of which is incorporated herein by reference in its entirety.

One skilled in the art will readily appreciate that any mouse or mouse cell (eg, a mouse ES cell modified in a reverse chimeric fashion) can be modified to include a modified Ig V segment as described herein. will be. In some embodiments, described herein is a genetically modified non-human animal having a genome, e.g., a germline genome comprising:

An endogenous immunoglobulin heavy chain variable region comprising a modified hV _H gene segment, a human D _H gene segment, and a human J _H gene segment of the present invention, wherein the immunoglobulin heavy chain variable region is operably linked to a constant region. immunoglobulin loci, and/or

An endogenous chain locus comprising an immunoglobulin light chain variable region comprising a modified V _L segment of the invention and a human J _L gene segment, wherein the immunoglobulin light chain variable region is operably linked to a constant region.

In some embodiments, a non-human animal, eg, a rodent, eg, a rat or mouse, has in its genome one or more endogenous V _H , D _H , and J _H segments at an endogenous immunoglobulin heavy chain locus and one or more human V _H , D _H , and J _H segments, wherein at least one human V _H , D _H , and J _H segment comprises a modified human V _H gene segment as described herein and an endogenous immunoglobulin is operably linked to a heavy chain gene; Optionally the unrearranged or rearranged human V _L and human J _L segments are selected from a non-human, eg, a rodent, eg, a mouse or rat, eg, at an endogenous non-human light chain locus, or operably linked to, for example, a human immunoglobulin light chain constant ( _CL ) region gene.

In certain embodiments, the genetically modified non-human animal has, in its genome, e.g., a germline genome, one or more unrearranged human immunoglobulins comprising a modified Ig V gene segment as described herein. One or more unrearranged human immunoglobulin variable regions comprising an immunoglobulin locus (exogenous or endogenous) containing an immunoglobulin variable region comprising variable region gene segments and an immunoglobulin constant region comprising immunoglobulin constant region genes; The gene segment is operably linked to an immunoglobulin constant region gene.

Generally, a genetically modified immunoglobulin locus comprises an immunoglobulin variable region (including immunoglobulin variable region gene segments) operably linked to an immunoglobulin constant region. In some embodiments, the genetically modified immunoglobulin locus comprises one or more human unrearranged immunoglobulin heavy chain comprising a modified Ig V _H gene segment as described herein operably linked to a heavy chain constant region gene. variable region gene segments. In some embodiments, the genetically modified immunoglobulin locus is a human unrearranged immunoglobulin variable region κ comprising a modified Ig Vκ gene segment as described herein operably linked to a κ chain constant region gene. contains gene segments. In some embodiments, the genetically modified immunoglobulin locus is a human unrearranged immunoglobulin variable region λ gene comprising a modified Ig Vλ gene segment as described herein operably linked to a κ chain constant region gene. contains fragments. In some embodiments, the genetically modified immunoglobulin locus is a human unrearranged immunoglobulin variable region λ comprising a modified Ig Vλ gene segment as described herein operably linked to a λ chain constant region gene. contains gene segments.

In certain embodiments, the non-human animal is an unrearranged human (localized) animal comprising an engineered modified Ig V _H gene segment as described herein operably linked to an endogenous heavy chain constant region at the endogenous heavy chain locus. An immunoglobulin heavy chain variable region, wherein the immunoglobulin variable region contains one or more unrearranged human Ig heavy chain variable region gene segments. In some embodiments, the one or more unrearranged human Ig variable region gene segments are modified Ig V _H gene segments, one or more immunoglobulin heavy chain diversity (D _H ) segments, and one or more immunoglobulin heavy chain segments as described herein. join (J _H ) segments (optionally one or more unrearranged human J _H segments). In some embodiments, a modified Ig V _H gene segment as described herein is the only Ig V _H segment present in a heavy chain variable region. In some embodiments, the unrearranged human Ig gene segments include all functional human _DH gene segments. In some embodiments, the unrearranged human Ig gene segments include all functional human _JH gene segments. Exemplary variable regions comprising Ig heavy chain gene segments are described, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and supplemental information, which is incorporated herein by reference in its entirety.

In some embodiments, a non-human animal provided herein comprises, at the endogenous heavy chain locus, a restricted unrearranged human (localized) heavy chain variable region operably linked to an endogenous heavy chain constant region comprising at least a non-human IgM gene. wherein the restricted unrearranged human (localized) heavy chain variable region is a single human _VH gene segment (eg, a single modified Ig _VH gene segment as described herein), a plurality of _DH gene segments ( eg, a human _DH gene segment) and a plurality of _JH gene segments (eg, a human _JH gene segment), wherein the restricted immunoglobulin heavy chain loci are capable of rearrangement and a plurality of distinct rearrangements wherein each rearrangement is derived from a single human _VH gene segment, one of the _DH segments and one of the _JH segments, wherein each rearrangement encodes a different heavy chain variable domain (e.g. As described, eg, in US Patent Publication No. 20130096287, which is incorporated herein by reference in its entirety). In some embodiments, the single modified human V _H gene segment is V _H 1-2 or V _H 1-69.

In certain embodiments, the non-human animal has an unrearranged human (localized) immunoglobulin light chain variable region comprising a modified Ig V _L segment as described herein operably linked to an endogenous light chain constant region. contains at the endogenous light chain locus. In some embodiments, the unrearranged human (localized) immunoglobulin light chain variable region contains unrearranged human Ig κ variable region gene segments. In some embodiments, the unrearranged human (localized) immunoglobulin variable region comprises one or a plurality of unrearranged human VK segments, which may include modified Ig VK segments not described herein, and one or more Contains unrearranged human _JK segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segments include all human Jκ segments. In some embodiments, the immunoglobulin variable region gene segments include four functional VK segments and all human JK segments. In some embodiments, the immunoglobulin variable region gene segments include 16 functional VK segments and all human JK segments (eg, all functional human VK segments and JK segments). In some embodiments, the unrearranged human immunoglobulin variable region gene segments include all human VK segments and all human JK segments. Exemplary variable regions comprising Ig κ gene segments are described, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 1 11:5147-52 and supplemental information, which is incorporated herein by reference in its entirety.

In some embodiments, a restricted unrearranged human (localized) light chain variable region operably linked to an endogenous light chain constant region is such that the unrearranged human (localized) light chain variable region comprises no more than two human V _L gene segments and characterized in that it comprises a plurality of J _L gene segments (e.g., a double light chain mouse as described in U.S. Patent No. 9,796,788, incorporated herein by reference in its entirety, or DLC), and no more than two human One of the V _L gene segments may be a modified Ig V _L segment as described herein. In some embodiments, the V _L gene segment is a VK gene segment. In some embodiments, the V _L gene segment is a Vλ gene segment. In some embodiments, the Vκ gene segments are IGKV3-20 and IGKV1-39. In some embodiments, a non-human animal comprises exactly two unrearranged human Vκ gene segments and five unrearranged human Jκ gene segments operably linked to a mouse light chain constant region at a mouse endogenous κ light chain locus; , optionally two unrearranged human VK gene segments are a human VK1-39 gene segment and a human VK3-20 gene segment, and the five unrearranged human JK gene segments are a human JK1 gene segment, a human JK2 gene segment, A human JK3 gene segment, a human JK4 gene segment, and a human JK5 gene segment, wherein the unrearranged human kappa light chain gene segment can be rearranged to encode a human variable domain of an antibody; It does not contain endogenous endogenous Vκ gene segments that can be rearranged to form globulin light chain variable regions.

In some embodiments, an unrearranged human (localized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region, including a modified Igλ segment as described herein, is an unrearranged human Igλ segment. Contains variable region gene segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise a plurality of human Vλ segments and one or more human Jλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise one or more human Vλ segments and one or more human Jλ segments and one or more human Cλ constant region sequences. In some embodiments, the unrearranged human immunoglobulin variable region gene segments include all human Vλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segments include all human Jλ segments. Exemplary variable regions comprising Ig λ gene segments are provided, for example, in US Pat. Nos. 9,035,128 and 6,998,514, each of which is incorporated herein by reference in its entirety. In some embodiments, an unrearranged human (localized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region comprises (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and ( c) one or more human Cλ gene segments, wherein (a) and (b) are operably linked to (c) and an endogenous (eg, rodent) Cλ gene segment, wherein the endogenous immunoglobulin λ light chain locus is one further comprising one or more rodent immunoglobulin λ light chain enhancers (Eλ), and one or more human immunoglobulin λ light chain enhancers (Eλ), optionally three human Eλs.

In certain embodiments, an unrearranged human (localized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region is a human derived from Vλ and Jλ gene segments in which a non-human animal is fused with an endogenous κ constant domain. An unrearranged human Igλ variable region gene segment operably linked to an endogenous (e.g., rodent, eg, rat or mouse) Cκ gene, such as to express an immunoglobulin light chain comprising a λ variable domain sequence, e.g. eg, modified Ig λ segments as described herein (see, eg, US Pat. No. 9,226,484, incorporated herein by reference in its entirety).

In some embodiments, a humanized immunoglobulin κ light chain locus, e.g., a humanized immunoglobulin endogenous κ locus, is located upstream of (e.g., operative) a Cλ gene that may replace an endogenous Cλ gene. one or more human Vλ gene segments (eg, a modified human Vλ segment as described herein) and one or more human Jλ gene segments (possibly linked). In some embodiments, the Cλ gene is a rodent (eg, rat or mouse) Cλ gene. In some embodiments, the Cλ gene is a mouse Cλ1 gene. In some embodiments, the Cλ gene comprises one or more human Cλ genes. In some embodiments, one or more human Jλ gene segments and one or more Cλ genes of such a humanized immunoglobulin κ light chain locus are in a Jλ-Cλ cluster. In some embodiments, the genetically modified rodent (eg, rat or mouse) is homozygous for this humanized immunoglobulin κ light chain locus. In some embodiments, the genetically modified rodent (eg, rat or mouse) is heterozygous for this humanized immunoglobulin κ light chain locus. In some embodiments, a genetically modified rodent (eg, rat or mouse) comprising such a humanized immunoglobulin κ light chain locus produces an antibody comprising, among other things, a λ light chain, each λ light chain comprising, e.g. eg, a human λ light chain variable domain operably linked to a human λ light chain constant domain in response to antigenic stimulation.

In some embodiments, an immunoglobulin variable region comprising an unrearranged human immunoglobulin variable region gene segment also comprises a human immunoglobulin variable region intergenic sequence. In some embodiments, an immunoglobulin variable region comprises non-human (eg, rodent, rat, mouse) Ig variable region intergenic sequences. In some embodiments, the intergenic sequence is from an endogenous species.

In some embodiments, the immunoglobulin variable region is a rearranged light chain variable region (universal light chain variable region). In some embodiments, the rearranged Ig light chain variable region gene is a human rearranged Ig light chain variable region gene. Exemplary rearranged Ig light chain variable regions are described in, eg, U.S. Patent Nos. 9,969,814; 10,130,181 and 10,143,186 and U.S. Patent Publication Nos. 20120021409, 20120192300, 20130045492, 20130185821, 20130302836, and 20150313193, each of which hereby in its entirety by reference integrated into In some embodiments, non-human organisms comprising a universal light chain variable region (“universal light chain” organisms) are used to produce bispecific antibodies. In some embodiments, the consensus light chain coding sequence comprises a single rearranged human immunoglobulin light chain VK/JK sequence operably linked to an endogenous light chain constant region. In some embodiments, a single rearranged human immunoglobulin light chain VK/JK sequence is modified to encode an anchor as described herein operably linked to a universal light chain. In some embodiments, the single rearranged human immunoglobulin light chain VK/JK sequence is (i) a human VK1-39/JK5 sequence comprising a human VK1-39 gene segment fused to a human JK5 gene segment, or (ii) a human VK1-39/JK5 sequence. A human VK3-20/JK1 sequence comprising a human VK3-20 gene segment fused to a JK1 gene segment.

In some embodiments, an immunoglobulin variable region is a light and/or heavy chain immunoglobulin variable region comprising insertions and/or substitutions of histidine codons designed to introduce pH dependent binding properties to antibodies produced in such non-human organisms. In some such embodiments, histidine codons are inserted and/or substituted into the nucleic acid sequence encoding CDR3. A variety of such light and/or heavy chain immunoglobulin loci are described in U.S. Patent Nos. 9,301,510; 9,334,334; and 9,801,362 and US Patent Application Publication No. 20140013456, each of which is incorporated herein by reference in its entirety. In some embodiments, a histidine modified rearranged human (localized) light chain variable region operably linked to an endogenous light chain constant region comprises one rearranged human immunoglobulin light chain variable region gene comprising human VK and JK segment sequences. wherein optionally the VK segment sequence is derived from a human VK1-39 or VK3-20 gene segment, and optionally one rearranged human immunoglobulin light chain variable region gene sequence (according to IMGT numbering) 105, 106 , 107, 108, 109, 111, and combinations thereof, and a histidine codon expressed at a position selected from the group consisting of at least one non-histidine codon of the Vκ segment sequence. In some embodiments, a histidine-modified unrearranged human (localized) heavy chain variable region operably linked to an endogenous heavy chain constant region comprises substitution of a histidine codon with at least one non-histidine codon or insertion of at least one histidine codon. An unrearranged human (localized) immunoglobulin heavy chain variable gene sequence comprising (eg, in a modified (human) V _H gene segment as described herein) in a complementarity determining region 3 (CDR3) coding sequence include In some embodiments, the unrearranged human (localized) immunoglobulin heavy chain variable gene sequence is an unrearranged human V _H (eg, a modified (human) V _H segment as described herein), a rearranged unrearranged human _DH or synthetic _DH , and unrearranged human _JH gene segments, optionally including unrearranged human _VH segments herein (e.g., modified (human modified as described herein) ) V _H segment) comprises substitution of histidine codon with at least one non-histidine codon or insertion of at least one histidine codon. In some embodiments, a histidine modified unrearranged human (localized) light chain variable region operably linked to an endogenous heavy chain constant region comprises unrearranged V _L and unrearranged J _L gene segments. In some embodiments, a histidine modified unrearranged human (localized) light chain variable region comprises no more than two unrearranged human V _L (eg, no more than two Vκ gene segments) and one or more unrearranged human V L (eg, no more than two Vκ gene segments) human J _L (e.g., Jκ) gene segment(s), wherein each of no more than two human V _L gene segments is a substitution of a histidine codon with at least one non-histidine codon and an insertion of at least one histidine codon. is included in the CDR3 coding sequence. In some embodiments, the no more than two human VK gene segments are human VK1-39 and VK3-20 gene segments, each of which comprises one or more substitutions of a histidine codon and a non-histidine codon, wherein the human VK and JK gene segments wherein the human Vκ and Jκ gene segments encode a human light chain variable domain comprising one or more histidines at positions selected from the group consisting of 105, 106, 107, 108, 109, 111 (according to IGMT numbering); , one or more histidines are derived from one or more substitutions.

In some embodiments, an immunoglobulin constant region comprises a heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is a human heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is of endogenous species origin. In some embodiments, the heavy chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the constant region gene is a mixture of human and non-human sequences. For example, in some embodiments, the constant region gene encodes a human CH1 region and a non-human (eg, from an endogenous species, mouse, rat) CH2 and/or CH3 region. In some embodiments, the heavy chain constant region gene is a Cμ, Cδ, Cγ (Cγl, Cγ2, Cγ3, Cγ4), Cα or Cε constant region gene. In some embodiments, the constant region gene is an endogenous constant region gene. In some embodiments, the constant region gene encodes a CH1 region mutated such that a non-human animal expresses a heavy chain only antibody (see, eg, US Pat. No. 8,754,287, US Patent Application Publication No. 2015/0289489). and each of which is incorporated herein by reference in its entirety). For example, in some embodiments where the goal is to generate a heavy chain for making a bispecific antibody (in a universal or dual light chain organism), the Fc domain of the heavy chain facilitates heavy chain heterodimer formation and/or heavy chain homologous Include modifications to inhibit mer formation. Such modifications are described in, for example, U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228 and 8,679,785, and US Patent Publication No. 2013/0195849, each of which is incorporated herein by reference in its entirety.

In some embodiments, an immunoglobulin constant region comprises a light chain constant region gene. In some embodiments, the light chain constant region gene is a κ constant region gene. In some embodiments, the light chain constant region gene is a λ constant region gene. In some embodiments, the light chain constant region genes are of endogenous species origin. In some embodiments, the light chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the light chain constant region gene is a mixture of human and non-human sequences.

In some embodiments, an immunoglobulin variable region comprising an immunoglobulin constant region gene and a human variable region gene segment to which the variable region gene segment is operably linked is located at an endogenous immunoglobulin locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous heavy chain locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous κ locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous λ locus. In some embodiments, the constant region gene to which human variable region gene segments are operably linked is an endogenous constant region gene.

In some embodiments, one or more endogenous immunoglobulin loci or portions of one or more endogenous loci (eg, variable regions and/or constant regions) within the genome of a non-human animal provided herein are inactivated. Endogenous immunoglobulin variable region loci and portions thereof can be obtained by deleting the locus or portion thereof from the genome of an organism, by substituting a locus or portion thereof with a different nucleic acid sequence, by replacing a gene or portion thereof with another location in the genome of a non-human organism. It can be inactivated using any method known in the art, including but not limited to inverting or displacing a portion thereof. In some embodiments, inactivation of a locus is only partial inactivation. In some embodiments, the variable region of the locus is inactivated but the constant region remains functional (eg, because the constant region is operably linked to a non-endogenous variable region gene segment).

In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin heavy chain locus. In some embodiments, the endogenous immunoglobulin heavy chain locus or portion thereof is inactivated by deletion, substitution, displacement, and/or inversion of at least a portion of the endogenous variable region of the endogenous heavy chain locus. In some embodiments, at least a portion of the variable region of the endogenous heavy chain locus that is deleted, substituted, displaced and/or inverted comprises a J segment of the variable region. In some embodiments, the endogenous immunoglobulin heavy chain locus or portion thereof is inactivated by deletion, substitution, displacement, and/or inversion of at least a portion of the endogenous constant region of the endogenous heavy chain locus. In some embodiments, at least a portion of the constant region of the endogenous heavy chain locus that is deleted, substituted, displaced and/or inverted comprises a Ομ gene of the endogenous constant region.

In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin κ chain locus. In some embodiments, the endogenous immunoglobulin κ chain locus or portion thereof is inactivated by deletion, substitution, displacement, and/or inversion of at least a portion of the endogenous variable region of the endogenous κ chain locus. In some embodiments, at least a portion of the variable region of the endogenous κ chain locus that is deleted, substituted, displaced and/or inverted comprises a J segment of the variable region. In some embodiments, the endogenous immunoglobulin κ chain locus or portion thereof is inactivated by deletion, substitution, displacement, and/or inversion of at least a portion of the endogenous constant region of the endogenous κ chain locus. In some embodiments, at least a portion of the constant region of the endogenous κ chain locus that is deleted, substituted, displaced and/or inverted comprises a CK gene of the endogenous constant region.

In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin λ chain locus. In some embodiments, the endogenous immunoglobulin λ chain locus or portion thereof is inactivated by deletion, substitution, displacement, and/or inversion of at least a portion of the endogenous variable region of the endogenous λ chain locus. In some embodiments, at least a portion of at least one V-J-C gene cluster within an endogenous λ chain locus is deleted, substituted, displaced, and/or inverted. In some embodiments, the endogenous immunoglobulin λ chain locus or portion thereof is inactivated by deletion, substitution, displacement, and/or inversion of at least a portion of the endogenous constant region of the endogenous λ chain locus. In some embodiments, at least a portion of the constant region of the endogenous λ chain locus that is deleted, substituted, displaced and/or inverted comprises the C gene of the endogenous constant region.

In various embodiments, modification of immunoglobulin loci does not affect fertility of the non-human animal. In some embodiments, the heavy chain locus comprises a functional gene, e.g., an endogenous ADAM6a gene, an ADAM6b gene, or both, and the genetic modification affects the expression and/or function of the endogenous ADAM6a gene, ADAM6b gene, or both. does not affect In some embodiments, the genome of the genetically modified non-human animal further comprises an ectopically located functional gene, eg, an endogenous ADAM6a gene, an ADAM6b gene, or both. Exemplary non-human animals expressing exogenous ADAM6a and/or ADAM6b are described in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety.

In some embodiments, the genetically modified non-human animal further comprises and expresses an exogenous terminal deoxytinucleotidyl transferase (TdT) to increase antigen receptor diversity. Exemplary non-human animals expressing exogenous TdT are described in PCT Publication WO 2017210586, which is incorporated herein by reference in its entirety.

In some embodiments, the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin k light chain transcriptional control element, an immunoglobulin l light chain transcriptional control element, or any combination thereof. do.

In some embodiments, the genome of a provided non-human animal comprises one or more human immunoglobulin heavy chain and/or light chain genes (e.g., U.S. Patent No. 8,502,018; U.S. Patent No. 8,642,835; U.S. Patent No. 8,697,940; U.S. Patent No. 8,791,323; and See U.S. Patent Application Publication Nos. 2013/0096287 A1 and 2018/0125043 A1; and PCT Publication No. WO2019/113065, each of which is incorporated herein by reference in its entirety. Alternatively, recombinant nucleic acid molecules can be introduced into embryonic stem cells of other modified strains, such as, for example, the VELOCIMMUNE® strain (see, eg, U.S. Patent No. 8,502,018 or U.S. Patent No. 8,642,835; incorporated herein by reference). In some embodiments, non-human animals as described herein can be constructed by introducing a targeting vector as described herein into cells from a transformed strain. To provide an example, targeting vectors as described herein can be introduced into non-human animals as described in U.S. Patent Nos. 8,642,835 and 8,697,940; This patent is incorporated herein by reference in its entirety, wherein the non-human animal expresses an antibody having fully human variable regions and mouse constant regions. In some embodiments, a non-human animal as described herein is engineered to further comprise human immunoglobulin genes (variable and/or constant region genes). In some embodiments, a non-human animal described herein comprises a modified Ig V segment, as described herein, and genetic material from a heterologous species (eg, human), wherein the genetic material is Encodes, in whole or in part, one or more human heavy and/or light chain variable regions.

Non-human animals as described herein may be prepared as described above or using methods known in the art to include additional human or humanized genes, often depending on the intended use of the non-human animal. The genetic material of such additional human or humanized genes is known in the art through further alteration of the genome of genetically modified cells (eg, embryonic stem cells) as described above, or by using other genetically modified lines as desired. can be introduced through established breeding techniques.

For example, as described herein, the genome of a non-human animal comprising a modified Ig V segment (e.g., through crossbreeding or multiple gene targeting strategies) can be generated in US Patent Application Publication No. 2011-0195454 A1 No., No. 2012-0021409 A1, No. 2012-0192300 A1, No. 2013-0045492 A1, No. 2013-0185821 A1, No. 2013-0198880 A1, No. 2013-0302836 A1, No. 2015-0059009 A1 ; further comprising one or more variants described in WO 2011/097603, WO 2012/148873, WO 2013/134263, WO 2013/184761, WO 2014/160179, WO 2014/160202; All of which are incorporated herein by reference in their entirety.

A transgenic founder non-human animal has an anchor-modified anchor-modified amino acid comprising an amino acid corresponding to a receptor-binding portion of a non-immunoglobulin polypeptide that binds a cognate receptor and/or the presence of a modified Ig V segment in its genome. It can be identified based on the expression of the antibody. The transgenic founder non-human animal can then be used to breed the modified Ig V segment with additional non-human animals to generate a series of non-human animals each having one or more copies of the modified Ig V segment. can In addition, the transgenic non-human animal with the modified Ig V segment can be further crossed with other transgenic non-human animals with other transgenes (eg, human immunoglobulin genes) as desired.

Transgenic non-human animals can also be produced to contain selected systems capable of regulating or inducing expression of the transgene. An exemplary system is the Cre/ lox P recombinase system of bacteriophage P1 (see, e.g., Lakso, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236, incorporated herein by reference in its entirety). included) and the FLT/Frt recombinase system of S. cerevisiae (O'Gorman, S. et al. 1991, Science 251:1351-1355; incorporated herein by reference in its entirety). Such animals can be, for example, one transgenic animal containing a transgene containing a selected modification (eg, a modified Ig V segment) and a transplant encoding a recombinase (eg, Cre recombinase). It can be provided through the construction of a “ double ” transgenic animal by crossing another transgenic animal that contains the gene.

Examples using modified Ig v segments in mice (i.e., mice with modified human V _H segment, D _H , and J _H gene segments, all operably linked to one or more murine heavy chain constant region genes) Although discussed extensively herein, other non-human animals comprising modified Ig v segments are also provided. Such non-human animals include, for example, mammals such as mice, rats, rabbits, pigs, cows (eg cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, any animal that can be genetically modified to express an anchor-modified immunoglobulin as described herein, including ferrets, primates (eg, marmosets, rhesus monkeys), and the like. For example, for non-human animals where suitable genetically modifiable ES cells are not readily available, other methods for producing non-human animals, including genetic modification, are employed. Such methods include, for example, modifying a non-ES cell genome (eg, a fibroblast or induced pluripotent cell) and delivering the genetically modified genome to a suitable cell, such as an enucleated oocyte. employing somatic cell nuclear transfer (SCNT) for embryo transfer, and impregnating the modified cells (eg, modified oocytes) into a non-human animal under conditions suitable to form an embryo.

Methods for modifying non-human animal genomes (eg, pigs, cows, rodents, chickens, etc.) include, for example, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs) ), or employing the Cas protein (ie, the CRISPR/Cas system) to modify the genome to include the modified Ig V segment described herein. Guidance on how to modify the germline genome of non-human animals can be found, for example, in US Patent Application Publication Nos. 2015-0376628 A1, US 2016-0145646 A1 and US 2016-0177339 A1; Its entirety is hereby incorporated by reference.

In some embodiments, a non-human animal as described herein is a mammal. In some embodiments, a non-human animal as described herein is, for example, a small mammal of the suborder Dipodoidea or Muroidea that jumps. In some embodiments, a genetically modified animal as described herein is a rodent. In some embodiments, a rodent as described herein is selected from mouse, rat and hamster. In some embodiments, a rodent as described herein is selected from the suborder Muridae. In some embodiments, a genetically modified animal as described herein is a member of the family Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and Mice, voles), Muridae (real mice and rats, desert mice, spiny mice, mane mice), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice ), Platacanthomyidae (eg, Spiny Dormouse), and Spalacidae (eg, Moles, Bamboo Rats, and Northeast). In some embodiments, a genetically modified rodent as described herein is selected from a genuine mouse or rat (muridae), gerbil, spiny mouse, crested rat. In some embodiments, a genetically modified mouse as described herein is selected from a member of the family Muridae. In some embodiments, a non-human animal as described herein is a rodent. In some embodiments, a rodent as described herein is selected from mouse and rat. In some embodiments, a non-human animal as described herein is a mouse.

In some embodiments, a non-human animal as described herein is C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/ 10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, a mouse of the present invention is 129P1, 129P2, 129P3, 129X1, 129S1 (eg, 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129/SvJae, 129S6 (129 /SvEvTac), 129S7, 129S8, 129T1, 129T2 (e.g., Festing et al., 1999, Mammalian genome 10:836; Auerbach, W. et al., 2000, Biotechniques 29 (5 ): 1024-1028, 1030, 1032; incorporated herein by reference in its entirety). In some embodiments, a genetically modified mouse as described herein is a mix of the aforementioned 129 strain and the aforementioned C57BL/6 strain. In some embodiments, a mouse as described herein is a mixture of the aforementioned 129 strains, or a mixture of the aforementioned BL/6 strains. In some embodiments, the 129 strain of a mix as described herein is a 129S6 (129/SvEvTac) strain. In some embodiments, a mouse as described herein is a BALB strain, eg, a BALB/c strain. In some embodiments, a mouse as described herein is a mix of a BALB strain and other strains described above.

In some embodiments, a non-human animal as described herein is a rat. In some embodiments, a rat as described herein is a Wistar rat, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. Agouti). In some embodiments, a rat strain as described herein is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fisher, F344, F6, and Arc Agouti.

Anchor-Modified Immunoglobulins and Methods of Producing Anchor-Modified Immunoglobulins

Several in vitro and in vivo techniques have been developed for the production of antibody-based therapeutics. In particular, in vivo techniques include random incorporation into an animal's genome (see, eg, U.S. Patent No. 5,569,825, incorporated herein by reference in its entirety) or operably linked with the animal's endogenous immunoglobulin constant region to generate endogenous Transgenics containing human immunoglobulin genes precisely located at immunoglobulin loci (see U.S. Patent Nos. 8,502,018; 8,642,835; 8,697,940; and 8,791,323; each of which is incorporated herein by reference in its entirety) The production of animals (ie rodents) is characterized. Both approaches have been productive in generating promising antibody therapeutic candidates for use in humans. In addition, both approaches have advantages over in vitro approaches in that antibody candidates are selected from the antibody repertoire generated in vivo , and affinity and specificity for antigens are selected within the internal environment of the host's immune system. include In this way, the antibody binds to its naturally presented antigen (within the relevant biological epitope and surface) rather than to artificial environments or in silico predictions that can accompany in vitro techniques. Despite the robust antibody repertoire produced from in vivo techniques, antibodies against complex (eg viruses, channel polypeptides) or cytoplasmic antigens remain challenging. In addition, generating antibodies against polypeptides that share a high degree of sequence identity between species (eg, humans and mice) remains a challenge due to immune resistance.

Thus, the present invention, among other things, allows somatic hypermutation of a larger repertoire of immunoglobulins capable of recognizing a cognate receptor and/or contributing to the avidity of the molecule through its own affinity for the cognate receptor, thus making the immunoglobulin of interest. Recognition is based on the construction of in vivo systems characterized by the production of antibodies with anchors that help anchor to the antigen (eg, the anchor's cognate receptor) and increase its affinity for the antigen.

A provided non-human animal can be used to make human antibodies, wherein the human antibodies comprise variable domains derived from one or more variable region nucleic acid sequences encoded by the genetic material of cells of the non-human animal described herein. do. For example, a given non-human animal is immunized with an antigen of interest (eg, a receptor cognate to an anchor) for a time and under conditions sufficient for the non-human animal to mount an immune response to the antigen of interest. . An antibody is isolated from a non-human animal (e.g., one or more cells, such as one or more B cells) and is tested for, eg, affinity, specificity, epitope mapping, ability to block ligand-receptor interactions, inhibitory receptor activation, etc. characterized using a variety of analytical measures, such as In some embodiments, an antibody produced by a given non-human animal comprises one or more human variable domains derived from one or more human variable region nucleotide sequences isolated from the non-human animal.

The non-human animals described herein provide a source of biological material (eg, cells) and improved in vivo systems that produce human antibodies useful for a variety of assays. In some embodiments, provided non-human animals are used to develop therapeutics that target one or more receptors of a ligand receptor pair as described herein. In some embodiments, a provided non-human animal is used to identify, screen and/or develop candidate therapeutics (eg, antibodies, etc.) that bind to one or more G-protein-coupled receptor (GPCR) polypeptides. In some embodiments, a provided non-human animal expresses one or more receptor tyrosine kinases, one or more human GPCR polypeptides, one or more Notch receptors, one or more of CD28, CTLA4, and PD1, plexin receptors, LDLR, LILR, KIR, and integrins. , or to screen and develop candidate therapeutics (eg, antibodies, etc.) that block the activity of an NPR, eg, NPR3. In some embodiments, a provided non-human animal comprises one or more human GPCR polypeptides, one or more Notch receptors, one or more of CD28, CTLA4, and PD1, a plexin receptor, LDLR, LILR, KIR, and an integrin, or NPR, such as For example, it is used to determine the binding profile of antagonists and/or agonists of NPR3. In some embodiments, a provided non-human animal comprises one or more human GPCR polypeptides, one or more Notch receptors, one or more of CD28, CTLA4, and PD1, a plexin receptor, LDLR, LILR, KIR, and an integrin, or NPR, such as For example, to determine the epitope or epitopes of one or more candidate therapeutic antibodies that bind NPR3.

In some embodiments, a given non-human animal is used to determine the pharmacokinetic profile of an antibody. In some embodiments, one or more given non-human animals and one or more control or reference non-human animals receive one or more candidate therapeutic antibody at various doses (e.g., 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg/day). kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/mg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg or more), respectively. Candidate therapeutic antibodies can be administered via any desired route of administration, including parenteral and non-parenteral routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intrathecal, intraventricular, intracranial, intrapleural infusion routes or other routes of infusion. Non-parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, intravaginal, and intraocular. Administration can also be continuous infusion, topical administration, sustained release from an implant (gel, membrane, etc.), and/or intravenous infusion, eg using an intravenous fluid bag. Blood was sampled at various time points (e.g., 0 h, 6 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or longer) from non-human animals (humanized and control). A variety of assays, including but not limited to total IgG, anti-therapeutic antibody response, aggregation, and the like, can be used to determine the pharmacokinetic profile of an administered candidate therapeutic antibody using samples obtained from non-human animals described herein. can be performed for

In some embodiments, a provided non-human animal expresses an antibody, whereby cells, cell lines, and cell cultures are generated in binding and functional assays, particularly where the antagonist or agonist is specific for a human polypeptide sequence or epitope; Alternatively, it serves as a source of antibodies for use in assays for the binding or function of, for example, antagonists or agonists, if specific for a human polypeptide sequence or epitope that functions in ligand receptor interaction (binding). In some embodiments, the epitope bound to a candidate therapeutic antibody or siRNA can be determined using cells isolated from a given non-human animal.

In some embodiments, provided cells from non-human animals can be isolated and used as needed, or maintained in culture for many generations. In some embodiments, a provided cell from a non-human animal is immortalized (eg, through the use of a virus) and maintained in culture (eg, in subculture) indefinitely.

In some embodiments, non-human animals as described herein provide an in vivo system for the generation of antibody variants that bind human target antigens. Such variants include antibodies with the desired functionality, specificity, low cross-reactivity towards a common epitope shared by two or more human target antigens. In some embodiments, a given non-human animal is used to generate an antibody panel to generate a series of antibody variants that are screened for desired or improved functionality.

In some embodiments, non-human animals described herein provide an in vivo system for generating antibody libraries. Such libraries can be grafted onto different Fc regions based on desired functional group function and/or variable regions using techniques known in the art (e.g., site-directed mutagenesis, error-prone PCR, etc.). Provides a source for heavy and light chain variable region sequences that can be used as a source for affinity maturation of sequences.

kit

The present invention further provides a pack or kit comprising one or more containers containing at least one non-human animal, non-human cell, DNA fragment, and/or targeting vector as described herein. The kit may be used in any applicable method (eg, research method). Such container(s) may optionally be affixed with a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biologicals, which notice is: (a) made, used, and sold for human administration; Agency approvals for sale, (b) instructions for use, or both, or transfers of materials and/or biological products (eg, non-human animals or non-human cells described herein) between two or more entities. The governing contract is reflected.

Non-limiting examples are described below:

Example 1. A recombinant nucleic acid molecule comprising a modified immunoglobulin (Ig) variable (V) segment encoding an anchor-modified Ig polypeptide,

The modified Ig V segment herein refers to a nucleic acid sequence encoding an Ig signal peptide and framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment, or a variant thereof. A nucleic acid sequence encoding an anchor between nucleic acid sequences encoding

wherein the anchor modified Ig polypeptide is operatively linked to:

(i) an Ig signal peptide;

(ii) an anchor; and

(iii) comprises FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment, or variants thereof;

wherein the anchor comprises a receptor binding portion of a non-immunoglobulin polypeptide of interest that binds to a cognate receptor;

Optionally, wherein the nucleic acid molecule lacks any other V segments.

Example 2. The recombinant nucleic acid molecule of Example 1, wherein the Ig signal peptide is a signal peptide from a germline Ig V segment, or a variant thereof.

Example 3. The method according to Example 1 or 2, wherein the germline Ig V segment or variant thereof is a germline Ig heavy chain variable (V _H ) segment or variant thereof,

A modified Ig V segment is a combination of a nucleic acid sequence encoding an Ig signal peptide and framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _H segment or variant thereof. A modified Ig V _H segment comprising a nucleic acid sequence encoding an anchor between the encoding nucleic acid sequences;

Anchor modified Ig polypeptides into operative linkages:

(i) an Ig signal peptide;

(ii) an anchor; and

(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _H segment or variant thereof.

Example 4. The method according to any one of Examples 1 to 3, wherein the germline Ig V segment or variant thereof is a germline human (h) V _H 1-2 segment, a germline hV _H 1-3 segment, a germline hV _H 1-8 segments, germline hV _H segments 1-18, germline hV _H segments 1-24, germline hV _H segments 1-45, germline hV _H segments 1-46, germline hV _H segments 1-58; germline hV _H 1-69 segments, germline hV _H 2-5 segments, germline hV _H 2-26 segments, germline hV _H 2-70 segments, germline hV _H 3-7 segments, germline hV _H 3 segments -9 segments, germline hV _H segments 3-11, germline hV _H segments 3-13, germline hV _H segments 3-15, germline hV _H segments 3-16, germline hV _H segments 3-20, reproduction Line hV _H segment 3-21, germline hV _H segment 3-23, germline hV _H segment 3-30, germline hV _H segment 3-30-3, germline hV _H segment 3-30-5, germline hV _H 3-33 segment, germline hV _H 3-35 segment, germline hV _H 3-38 segment, germline hV _H 3-43 segment, germline hV _H 3-48 segment, germline hV _H 3-49 segment, germline hV _H 3-53 segment, germline hV _H 3-64 segment, germline hV _H 3-66 segment, germline hV _H 3-72 segment, germline hV _H 3-73 segment, germline hV _H 3-74 segments, germline hV _H 4-4 segments, germline hV _H 4-28 segments, germline hV _H 4-30-1 segments, germline hV _H 4-30-2 segments, germline hV _H Segment 4-30-4, germline hV _H 4-31 segment, germline hV _H 4-34 segment, germline hV _H 4-39 segment, germline hV _H 4-59 segment, germline hV _H 4-61 A recombinant nucleic acid molecule that is a segment, a germline hV _H 5-51 segment, a germline hV _H 6-1 segment, a germline hV _H 7-4-1 segment, a germline hV _H 7-81 segment, or a variant thereof.

Example 5. The method according to any one of Examples 1 to 4, wherein the germline Ig V segment or variant thereof is a germline hV _H 1-69 segment or variant thereof, optionally wherein the Ig signal peptide has the sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7 ), Recombinant nucleic acid molecules, including.

Example 6. The method of any one of Examples 3 to 5, with an operable connection and from 5' to 3':

(I) a modified Ig V _H segment;

(II) one or multiple Ig heavy chain diversity (D _H ) segments, and

(III) a recombinant nucleic acid molecule comprising one or a plurality of Ig heavy chain linking (J _H ) segments.

Example 7. In Example 6,

The one or plurality of Ig D _H segments of (II) comprises one, a plurality of, or all human Ig D _H segments;

A recombinant nucleic acid molecule, wherein the one or plurality of Ig J _H segments of (III) comprises one, a plurality of, or all human Ig J _H segments.

Example 8. The one or plurality of Ig D _H gene segments of (II) and the one or plurality of Ig J _H gene segments of (III) according to Example 6 or 7 are recombined and rearranged Ig D _H /J _H sequences are formed so that the recombinant nucleic acid molecule is 5' to 3' in an operative linkage.

Modified Ig V _H gene segments and

A recombinant nucleic acid molecule comprising a rearranged Ig D _H /J _H sequence.

Example 9. The modified Ig _{V H} gene segment _of Example 8 and the rearranged Ig D _H /J _H sequence are recombined to encode the anchor modified Ig heavy chain variable domain. /J forms _{the H} sequence;

wherein the anchor modified Ig heavy chain variable domain is operatively linked to:

(i) an Ig signal peptide;

(ii) an anchor; and

(iii) a recombinant nucleic acid molecule comprising FR1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _H /D _H /J _H sequence.

Example 10. The recombinant nucleic acid molecule according to any one of Examples 3 to 8, wherein the modified Ig V _H segment is an unrearranged modified Ig V _H gene segment.

Example 11. A recombinant nucleic acid molecule according to any one of Examples 6 to 10, further comprising a nucleic acid sequence encoding an Ig heavy chain constant region ( _CH ),

wherein the nucleic acid sequence encoding Ig _CH is

(I) a modified Ig V _H segment;

(II) one or multiple Ig D _H segments, and

(III) a recombinant nucleic acid molecule operably linked downstream of one or more Ig J _H segments.

Example 12. The nucleic acid sequence of Example 11, wherein the nucleic acid sequence encoding Ig _CH is an Igμ gene encoding an IgM isotype, an Igδ gene encoding an IgD isotype, an Igγ gene encoding an IgG isotype, an IgA isotype A recombinant nucleic acid molecule comprising an Igα gene encoding, and/or an Igε gene encoding an IgE isoform.

Example 13. A recombinant nucleic acid molecule according to any one of Examples 3 to 12 comprising a nucleic acid sequence encoding an anchor-modified Ig heavy chain, wherein the anchor-modified Ig heavy chain is operatively linked to:

(i) an Ig signal peptide;

(ii) an anchor;

(iii) an Ig heavy chain variable domain comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _H /D _H /J _H sequence, and

(iv) a recombinant nucleic acid molecule comprising an Ig _CH .

Example 14. The recombinant nucleic acid molecule according to any one of Examples 11 to 13, wherein the Ig _CH is a non-human Ig _CH .

Example 15. The recombinant nucleic acid molecule of Example 14, wherein the non-human Ig _CH is a rodent Ig _CH .

Example 16. The recombinant nucleic acid molecule of Example 15, wherein the non-human Ig _CH is a rat Ig _CH .

Example 17. The recombinant nucleic acid molecule of Example 15, wherein the non-human Ig _CH is a mouse Ig _CH .

Example 18. The method according to Example 1 or Example 2, wherein the germline Ig V gene segment or variant thereof is a germline Ig light chain variable (V _L ) segment or variant thereof,

The modified Ig V segment combines a nucleic acid sequence encoding an Ig signal peptide with framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _L segment or variant thereof. A modified Ig V _L segment comprising a nucleic acid sequence encoding an anchor between the encoding nucleic acid sequences;

Anchor modified Ig polypeptides into operative linkages:

(i) an Ig signal peptide;

(ii) an anchor; and

(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _L segment or variant thereof.

Example 19. The method of Example 18, from 5' to 3' with an operable connection:

(I) a modified Ig V _L segment, and

(II) a recombinant nucleic acid molecule comprising one or a plurality of Ig light chain linking (J _L ) segments.

Example 20. The modified Ig V _L segment of Example 19 and one or more Ig J _L segments are recombined to form a rearranged Ig V _L /J _L sequence encoding an anchor modified Ig light chain variable domain. do,

Anchor modified Ig light chain variable domains with an operative linkage:

(i) an Ig signal peptide;

(ii) an anchor; and

(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _L /J _L sequence.

Example 21. A recombinant nucleic acid molecule according to Example 19 or 20, further comprising a nucleic acid sequence encoding an Ig light chain constant region ( _CL ),

The nucleic acid sequence encoding Ig C _L is:

(I) modified Ig V _L segments and

(II) a recombinant nucleic acid molecule operably linked downstream of one or a plurality of Ig light chain binding (J _L ) segments.

Example 22. A recombinant nucleic acid molecule according to any one of Examples 18 to 21 comprising a nucleic acid sequence encoding an anchor-modified Ig light chain, wherein the anchor-modified Ig light chain is operatively linked to:

(i) an Ig signal peptide;

(ii) an anchor;

(iii) an Ig light chain variable domain comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _L /J _L sequence, and

(iv) a recombinant nucleic acid molecule comprising an Ig C _L.

Example 23. The recombinant nucleic acid molecule of Example 21 or 22, wherein the Ig C _L is a non-human Ig C _L.

Example 24. The recombinant nucleic acid molecule of Example 23, wherein the non-human Ig C _L is a rodent Ig C _L.

Example 25. The recombinant nucleic acid molecule of Example 23, wherein the non-human Ig C _L is a rat Ig C _L.

Example 26. The recombinant nucleic acid molecule of Example 23, wherein the non-human Ig C _L is a mouse Ig C _L.

Example 27. The method according to any one of Examples 18 to 26, wherein the germline Ig V _L segment or variant thereof is a germline Ig light chain variable kappa (Vκ) segment or variant thereof,

The modified Ig V segment comprises a nucleic acid sequence encoding a signal peptide and a framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 encoding a germline Ig Vκ segment or variant thereof. A modified Ig Vκ segment comprising a nucleic acid sequence encoding an anchor between the nucleic acid sequences;

Anchor modified Ig polypeptides into operative linkages:

(i) an Ig signal peptide;

(ii) an anchor; and

(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig Vκ segment or variant thereof.

Example 28. The method of Example 27, from 5' to 3' with an operable connection:

(I) modified Ig Vκ segments, and

(II) A recombinant nucleic acid molecule comprising one or a plurality of Ig light chain binding kappa (Jκ) segments.

Example 29. The recombinant nucleic acid molecule according to Example 28, further comprising a nucleic acid sequence encoding an Ig light chain constant kappa region (CK),

wherein the nucleic acid sequence encoding the Ig CK is:

(I) modified Ig Vκ segments, and

(II) a recombinant nucleic acid molecule operably linked downstream of one or more Ig Jκ segments.

Example 30. The method according to any one of Examples 18 to 26, wherein the germline Ig V _L segment or variant thereof is a germline Ig light chain variable lambda (Vλ) segment or variant thereof, thereby

The modified Ig V segment encodes a nucleic acid sequence encoding an Ig signal peptide and framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig Vλ segment or variant thereof. A modified Ig Vλ segment comprising a nucleic acid sequence encoding an anchor between nucleic acid sequences that

Anchor modified Ig polypeptides into operative linkages:

(i) an Ig signal peptide;

(ii) an anchor; and

(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig Vλ segment or variant thereof.

Example 31. The method of Example 30, from 5' to 3' with an operable connection:

(I) Modified Ig Vλ segments, and

(II) A recombinant nucleic acid molecule comprising one or a plurality of Ig light chain binding lambda (Jλ) segments.

Example 32. The recombinant nucleic acid molecule according to Example 31, further comprising a nucleic acid sequence encoding an Ig light chain constant lambda region (Cλ),

The nucleic acid sequence encoding Ig Cλ is:

(I) Modified Ig Vλ segments, and

(II) a recombinant nucleic acid molecule operably linked downstream of one or more Ig Jλ segments.

Example 33. The method of any one of Examples 1-32, wherein the anchor connects the receptor binding portion of the non-immunoglobulin polypeptide of interest to FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment, or variants thereof. A recombinant nucleic acid molecule comprising a linker.

Example 34. The recombinant nucleic acid molecule of Example 33, wherein the linker comprises the sequence GGGGS (SEQ ID NO: 5).

Example 35. The recombinant nucleic acid molecule according to any of Examples 1-34, wherein the anchor comprises a natriuretic peptide receptor (NPR) binding portion of a natriuretic peptide (NP).

Example 36. The recombinant nucleic acid molecule of Example 35, wherein the NPR binding portion of NP comprises the C-terminal tail of NP.

Example 37. The recombinant nucleic acid molecule of Example 35 or 36, wherein the NP is atrial natriuretic peptide (ANP).

Example 38. The recombinant nucleic acid molecule according to any one of Examples 1-37, wherein the anchor comprises the sequence NSFRY (SEQ ID NO: 3).

Example 39. The sequence presented as SEQ ID NO: 8 or a degenerate variant thereof according to any one of Examples 1 to 38, the sequence presented as SEQ ID NO: 10 or a degenerate variant thereof, SEQ ID NO: 11 which is a degenerate variant thereof, and SEQ ID NO: 12 or a degenerate variant thereof. A recombinant nucleic acid molecule comprising a sequence selected from the group consisting of:

Example 40. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-10 and 33-39, wherein the targeting vector, upon homologous recombination between the targeting vector and a non-human Ig heavy chain locus, has a targeted ratio -Adding 5' and 3' homology arms targeting the non-human Ig heavy chain locus such that the human Ig heavy chain locus contains the recombinant nucleic acid molecule in an operative linkage upstream of the non-human C _H at the non-human Ig heavy chain locus. optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus, and/or the non-human Ig heavy chain locus is a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V _H , D _H , and/or or a deletion of the J _H gene segment, or a combination thereof.

Example 41. The targeting vector of Example 40, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule displaces the non-human V _H segment at the non-human Ig heavy chain locus.

Example 42. The method of embodiment 40 or 41, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule is one or more non-human V _H segments at the non-human Ig heavy chain locus, all non-human Ig heavy chain loci -a targeting vector that substitutes the human D _H segment, and all non-human J _H segments.

Example 43. The method according to any one of Examples 40 to 42, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule produces one non-human V _H segment at the non-human Ig heavy chain locus. or a targeting vector that substitutes all but all non-human V _H segments, all non-human D _H segments, and all non-human J _H segments.

Example 44. The method according to any one of Examples 40 to 43, wherein upon homologous recombination between the vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus is transferred from the non-human Ig heavy chain locus to the non-human Ig heavy chain regulatory sequence. A targeting vector comprising a recombinant nucleic acid molecule in operative linkage.

Example 45. The targeting vector of any one of Examples 40-44, wherein the 5' homology arm comprises the sequence presented as SEQ ID NO: 11 and/or the 3' homology arm comprises the sequence presented as SEQ ID NO: 12.

Example 46. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-17 and 33-39, wherein the targeting vector, upon homologous recombination between the targeting vector and a non-human Ig heavy chain locus, 5' and 3' homology arms targeting the non-human Ig heavy chain locus such that the non-human Ig heavy chain locus contains a recombinant nucleic acid molecule in operable linkage to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus. further comprising, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or the non-human Ig heavy chain locus is a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V _H , D _H , and/or or a deletion of the J _H gene segment, or a combination thereof.

Example 47. The method of Example 46, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule is at least one non-human V _H segment, all non-human at the non-human Ig heavy chain locus A targeting vector that substitutes D _H gene segments, all non-human J _H gene segments, and one or more non-human C _H genes.

Example 48. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-2, 18-20, 27-28, 30-31, and 33-38, wherein the targeting vector is a targeting vector and a non-human Upon homologous recombination between the Ig light chain loci, the non-human Ig light chain locus such that the targeted non-human Ig light chain locus contains the recombinant nucleic acid molecule in an operative linkage upstream of the non-human Ig C _L at the non-human Ig light chain locus. optionally further comprising 5' and 3' homology arms targeting the locus, wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or the non-human Ig light chain locus is a human or humanized immunoglobulin light chain locus A targeting vector comprising a deletion of a variable region, endogenous Ig V _L and/or J _L gene segments, or a combination thereof.

Example 49. The targeting vector of Example 48, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule displaces the non-human V _L segment at the non-human Ig light chain locus.

Example 50. The method of Example 48 or Example 49, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule comprises at least one non-human V _L segment and all non-human J _L segments Substituting, targeting vector.

Example 51. The method according to any one of Examples 48 to 50, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule is obtained at the non-human Ig light chain locus to all non-human V _L segments and A targeting vector, substituting all non-human J _H segments.

Example 52. The method according to any one of Examples 48 to 51, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus is operable to the non-human Ig light chain regulatory sequence at the Ig light chain locus. A targeting vector comprising a recombinant nucleic acid molecule in linkage.

Example 53. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-2, 18-38, wherein the targeting vector upon homologous recombination between the targeting vector and a non-human Ig light chain locus, the targeted ratio - add 5' and 3' homology arms targeting the non-human Ig light chain locus such that the human Ig light chain locus contains a recombinant nucleic acid molecule in operable linkage to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or the non-human Ig light chain locus is a human or humanized immunoglobulin light chain variable region, an endogenous Ig V _L and/or J _L gene segment A targeting vector comprising a deletion of, or a combination thereof.

Example 54. The method of Example 53, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule is a non-human V _L segment, all non-human J _L segments at the non-human Ig light chain locus A targeting vector that replaces a gene segment, and a non-human C _L gene.

Example 55. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-2, 18-20, 27-28, and 33-38, wherein the targeting vector has homology between the targeting vector and the non-human Ig light chain κ locus. 5 targeting the non-human Ig light chain κ locus such that, upon recombination, the targeted non-human Ig light chain κ locus contains a recombinant nucleic acid molecule in an operative linkage upstream of the non-human Ig CK at the non-human Ig light chain κ locus. ' and 3' homology arms, optionally wherein the non-human Ig light chain κ locus is an endogenous rodent Ig light chain κ locus and/or the non-human Ig light chain κ locus is a human or humanized immunoglobulin light chain variable region. , deletion of endogenous Ig Vκ and/or Jκ gene segments, or a combination thereof.

Example 56. The targeting vector of Example 55, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule substitutes a non-human VK segment at the non-human Ig light chain κ locus.

Example 57. The method of Example 55 or Example 56, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule comprises at least one non-human Vκ segment and all non-human Vκ segments at the non-human Ig light chain κ locus. A targeting vector substituting the human Jκ segment.

Example 58. The method of any one of Examples 55 to 57, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule comprises all non-human Vκ segments at the non-human Ig light chain κ locus and all non-human Vκ segments at the non-human Ig light chain κ locus. -a targeting vector that substitutes for a human Jκ segment.

Example 59. The method according to any one of Examples 55 to 58, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus regulates the non-human Ig light chain κ locus at the Ig light chain κ locus. A targeting vector comprising a recombinant nucleic acid molecule in operative linkage to a sequence.

Example 60. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-2 and 18-29, wherein the targeting vector, upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, is targeted non-human 5' and 3' homology arms targeting the non-human Ig light chain κ locus such that the Ig light chain κ locus comprises a recombinant nucleic acid molecule in operable linkage to a non-human Ig light chain κ regulatory sequence at the Ig light chain κ locus Including, the targeting vector.

Example 61. The method of Example 60, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule is a non-human Vκ segment at the non-human Ig light chain κ locus, all non-human Jκ gene segments, and A targeting vector that substitutes for a non-human CK gene.

Example 62. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-2, 18-20, 30-31, and 33-38, wherein the targeting vector has homology between the targeting vector and the non-human Ig light chain λ locus. 5' targeting the non-human Ig light chain λ locus such that, upon recombination, the targeted non-human Ig light chain λ locus contains a recombinant nucleic acid molecule in an operative linkage upstream of the non-human Ig Cλ at the non-human Ig light chain locus. and a 3' homology arm, optionally wherein the non-human Ig light chain λ locus is an endogenous rodent Ig light chain λ locus and/or the non-human Ig light chain λ locus is a human or humanized immunoglobulin light chain variable region; A targeting vector comprising a deletion of endogenous Ig Vλ and/or Jλ gene segments, or a combination thereof.

Example 63. The targeting vector of Example 62, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule substitutes a non-human Vλ segment at the non-human Ig light chain λ locus.

Example 64. The method of Example 62 or Example 63, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule comprises at least one non-human Vλ segment and all non-human Vλ segments at the non-human Ig light chain locus. A targeting vector, substituting the Jλ segment.

Example 65. The method according to any one of Examples 62 to 64, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule comprises all non-human Vλ segments at the non-human Ig light chain λ locus and all non-human Vλ segments at the non-human Ig light chain λ locus. -a targeting vector that substitutes for the human Jλ segment.

Example 66. The method according to any one of Examples 62 to 65, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus regulates the non-human Ig light chain λ locus at the Ig light chain λ locus. A targeting vector comprising a recombinant nucleic acid molecule in operative linkage to a sequence.

Example 67. A targeting vector comprising the recombinant nucleic acid molecule of any one of Examples 1-2, 18-26, and 30-38, wherein the targeting vector, upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, targets 5' and 3' homology targeting the non-human Ig light chain λ locus to include a recombinant nucleic acid molecule with an operative linkage at the Ig light chain λ locus to a non-human Ig light chain λ regulatory sequence. A targeting vector, further comprising cancer.

Example 68. The method of Example 67, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule is a non-human Vλ segment at the non-human Ig light chain λ locus, all non-human Jλ gene segments, and A targeting vector that substitutes for a non-human Cλ gene.

Example 69. A non-human animal genome comprising the recombinant nucleic acid molecule of any one of Examples 1-39 or the targeting vector of any one of Examples 40-68, optionally wherein the non-human animal is a rodent, and optionally wherein the rodent is Non-human animal genome, either rat or mouse.

Example 70. The non-human animal genome of Example 69, wherein the recombinant nucleic acid is at an endogenous Ig locus of the non-human animal genome.

Example 71. A non-human animal or non-human comprising the recombinant nucleic acid molecule of any one of Examples 1-39, the targeting vector of any one of Examples 40-68, or the non-human animal genome of Examples 69 or 70 zooblast.

Example 72. The non-human animal or non-human animal cell of Example 71, wherein the recombinant nucleic acid molecule, targeting vector, or non-human animal genome is in the germline of the non-human animal or non-human animal cell.

Example 73. An in vitro method of modifying an isolated cell comprising introducing a recombinant nucleic acid molecule of any one of Examples 1-39 into the isolated cell.

Example 74. The in vitro method of Example 73, wherein introducing comprises contacting the cell with the targeting vector of any one of Examples 40-68.

Example 75. The in vitro method of Example 73 or Example 74, wherein the cell is a host cell.

Example 76. The in vitro method of Example 73 or Example 74, wherein the cells are embryonic stem (ES) cells.

Example 77. The in vitro method of any one of examples 73-76, wherein the cells are rodent cells, optionally the rodent cells are rat cells or mouse cells.

Example 78. A non-human animal embryo generated from the embryonic stem cells of Example 76.

Example 79. Non-human animals generated from the embryonic stem cells of Example 76

Example 80. A non-human animal comprising transplanting the ES cell or embryo comprising the ES cell of Example 76 into a suitable host and maintaining the host under suitable conditions while the ES cell or embryo develops into a viable progeny. manufacturing method.

Example 81. In the non-human animal of any one of Examples 71-72 and 79 or the non-human animal prepared according to the method of Example 80, the non-human animal compared to a control non-human animal:

(a) comparable numbers of mature B cells in the spleen;

(b) comparable numbers of kappa-positive B cells in the spleen;

(c) comparable numbers of lambda positive B cells in the spleen;

(d) comparable levels of serum IgG and/or

(e) non-human animals with comparable levels of serum IgM.

Example 82. In the non-human animal of any one of Examples 71-72, 79 and 81, or the non-human animal prepared according to the method of Example 80, the non-human animal has immunity comparable to that of the control non-human animal. -A non-human animal capable of responding.

Example 83. In the non-human animal of any one of Examples 71-72, 79 and 81-82 or prepared according to the method of Example 80, a plurality of antigen- A non-human animal comprising a binding protein, optionally:

wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or

The mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry, or

The mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, and the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry. .

Example 84. In the non-human animal of any one of Examples 71-72, 79 and 81-83, or the non-human animal prepared according to the method of Example 80, the non-human animal is a cognate of the non-immunoglobulin polypeptide of interest. A non-human animal, further comprising a receptor.

Example 85. In the non-human animal of any one of Examples 71 to 72, 79 and 81 to 84 or the non-human animal prepared according to the method of Example 80, each comprising an anchor-modified Ig polypeptide and the non- A non-human animal comprising a plurality of antigen-binding proteins that specifically bind to a cognate receptor of an immunoglobulin polypeptide, optionally:

wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or

The mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry, or

The mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, and the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry. .

Example 86. The non-human animal of any one of Examples 84-85, wherein the cognate receptor is a natriuretic peptide receptor (NPR).

Example 87. The non-human animal of any of examples 84-86, wherein each of the plurality of antigen-binding proteins comprises a KD of less than 1X10 ⁹ and/or a t½ greater than 30 minutes.

Example 88. The non-human animal of any one of Examples 84-87, wherein at least 15% of the plurality of antigen-binding proteins block binding of a cognate receptor to a non-immunoglobulin polypeptide of interest.

Example 89. The non-human animal of any one of Examples 84-88, wherein greater than 50% of the plurality of antigen-binding proteins bind to cognate receptors expressed on the cell surface.

Example 90. The non-human animal of any one of Examples 71-72, 79, and 81-89, or the non-human animal prepared according to the method of Example 80, wherein the non-human animal is a rodent, optionally wherein the rodent is a non-human animal, such as a rat or mouse.

Example 91. A method of producing an antigen-binding protein or obtaining a nucleic acid encoding it, the method comprising:

Immunizing a non-human animal of any one of Examples 71 to 72, 79, and 81 to 90, or a non-human animal prepared according to the method of Example 80, with an antigen;

enabling a non-human animal to produce an antigen-binding protein comprising an anchor-modified Ig polypeptide that binds an antigen or a nucleic acid encoding the same, and optionally:

wherein the mass of the antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or

The mass of the antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry, or

wherein the mass of the antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, and the mass of the antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry.

Example 92. The method of Example 91, further comprising recovering the antigen binding protein or nucleic acid encoding it from the non-human animal or non-human animal cells.

Example 93. The method of Example 92, wherein the non-human animal cell is a B cell or hybridoma.

Example 94. Non-human animal cells, recovered according to the method of Example 91.

Example 95. The non-human animal cell of Example 94, wherein the non-human animal cell is a B cell.

Example 96. The non-human animal cell of Example 94 or 95, wherein the B cell is a mouse B cell.

Example 97. A hybridoma cell comprising the non-human animal cell of any one of Examples 94-95 fused with a myeloma cell.

Example 98. The recombinant nucleic acid molecule of any one of Examples 1-39, the targeting vector of any one of Examples 40-68, the non-human animal genome of any one of Examples 69-70, and By a non-human animal or non-human animal cell prepared according to the method of any one of Examples 73-76 and 80, expressed by the non-human animal or non-human animal cell of any one of 81-90. An anchor-modified Ig polypeptide expressed or prepared according to the method of any one of Examples 91-93, optionally:

wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or

The mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry, or

The mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, and the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectroscopy. polypeptide.

Example 99. The anchor-modified Ig polypeptide of Example 98 comprising at its N-terminus the amino acid sequence set forth in SEQ ID NO: 3.

Other features of the above-described embodiments will become apparent in the course of the following description of exemplary embodiments, which are provided for purposes of illustration and are not intended to be limiting.

Example

The following examples are provided to explain to those skilled in the art how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise specified, temperatures are expressed in degrees Celsius and pressures are at or near atmospheric.

Example 1. Constructs of Immunoglobulin Variable Regions Containing ANP-Modified Immunoglobulin Variable Region Gene Segments

This non-limiting example illustrates the construction of targeting vectors for integrating anchor-modified immunoglobulin (Ig) variable region (V) gene segments into immunoglobulin variable regions of immunoglobulin loci. As described below, the coding sequence of the ANP C-terminal tail is placed in operably linked with an Ig V gene segment, and the modified Ig V segment is placed in operably linked with an Ig binding (J) gene segment, where appropriate, with an Ig It can be placed in operably linked with the diversity (D) gene segment, so upon V(D)J recombination, an antibody comprising an ANP C-terminal tail at the N-terminus of the immunoglobulin polypeptide chain is expressed.

Targeting vectors containing ANP-modified _VH gene segments for insertion into immunoglobulin heavy chain variable regions can be prepared using the VELOCIGENE® technology (e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., 2003, Nature Biotech. 21(6):652). -659; incorporated herein by reference) and molecular biology techniques known in the art. A non-limiting exemplary strategy for constructing a targeting vector using a sequence encoding the C-terminal tail of ANP is presented in FIGS. 1-2 .

Insertion of a donor DNA segment containing the C-terminal tail of ANP through a sequence encoding a peptide linker into the germline V _H 1-69 gene segment by a novel DNA synthesis method (Blue Heron Biotech, Bothell, Wash.) performed. 1 shows the “p466090” ANP-V _H 1-69 Cas9 donor fragment. The spectinomycin-resistance cassette “SPEC” was ligated into the EcoRI and AvrII sites of the donor plasmid p46609 to create plasmid p46685 ( FIG. 1 ). Shown in Figure 2 is a more detailed view of the resulting donor plasmid p46609.

The donor fragment was used to transform BAC clone VI504 (MAID6211). See Figure 2 . BAC clone VI504, 5' to 3', about 20 kb of 5' about 20 kb 5' mouse homology arm, mouse Adam6a gene “a”, Frt-Ub-Hyg-Frt cassette, and mouse Adam6b gene “b” about 15 kb I-CeuI-AscI fragment containing germline human V _H 1-69 gene, about 9 kb AscI-AsiSI fragment containing human D H and human J H genes, and about 60 kb fragment containing human D _H and human J _H genes, and mouse IgH intron It contains an approximately ˜8 kb 3′ mouse homology arm containing the enhancer (filled oval), the IgM switch region, and part of the mouse IgM gene ( FIG. 3 ).

In step 1, VI504 was digested in vitro with Cas9 complexed with a mixture of two gRNAs cleaving 5' and 3' of the germline V _H 1-69 gene. The resulting 3' and 5' ends overlap the ends of p46685 by 60 bp. Then, VI738 was prepared by assembling the XhoI fragment of p46685 with VI504 (MAID6211) by Gibson Assembly. In step 2, VI738 was digested with MreI to remove the spectinomycin-resistance cassette. The BAC was then repaired by zygote oligo-mediated Gibson Assembly, leaving a seamless junction (ΔMreI) to produce the final LTVEC VI748 (MAID6833). LTVEC was identical to VI504 except for the insertion of the ANP-G4S codon in VI748.

Precise assembly of donor fragments as described herein and targeting of the germline (GL) V _H 1-69 gene segment of BAC clone VI504 to the NP-modified V _H 1-69 gene segment as described herein Substitutions were sequenced throughout the construction of the targeting vector using the primers shown in Table 1 and confirmed by polymerase chain reaction.

step join Primer name primer sequence
(SEQ ID NO) Jxn PCR (bp) A. p46685 was prepared by EcoRI/AvrII linkage of pSVi0029 to p46609 Jxn PCR EcoRI 5' detection V _H 1-69 FR3 ACAGAAGTTCCAGGGCAGAG
(SEQ ID NO: 18) 302 3' upstream detection details TGTCCACTGGGTTCGTGCCTT
(SEQ ID NO: 19) Jxn PCR AvrII 5' downstream detection details CAGTATCAGCCCGTCATACTT
(SEQ ID NO: 20) 197 3' V _H 1-69 overlap detection TAACCCCTGTCATCTCCTC
(SEQ ID NO: 21) 1. Cas9/GA of VI504 (MAID6211) + p46685 = VI738 Cas9 5' DNA target (PAM) GGATCCTGGTTTAGTTAAAG(AGG)
(SEQ ID NO: 22) 5′ V _H 1-69 Cas9 crRNA GGAUCCUGGUUUAGUUAAAG GUUUUAGAGCUAUGCUGUUUUG
(SEQ ID NO: 23) 3' DNA target (PAM) GACAAAAACCCTGAGGGAGA(AGG)
(SEQ ID NO: 24) 3 V _H 1-69 Cas9 crRNA GACAAAAACCCUGAGGGAGA GUUUUAGAGCUAUGCUGUUUUG
(SEQ ID NO: 25) Jxn PCR _5′VH 1-69 5' upstream detection V _H 1-69 CTGTGAAATACCCTGCCTC
(SEQ ID NO: 26) 797 3' upstream detection details TGTCCACTGGGTTCGTGCCTT
(SEQ ID NO: 27) Jxn PCR _3′VH 1-69 5' downstream detection details CAGTATCAGCCCGTCATACTT
(SEQ ID NO: 28) 631 3' downstream detection M1116 (h70) CCCCCTCTTGCTCTCTTTCT
(SEQ ID NO: 29) 2. Deletion of MreI_Spec from VI738 by zygotic oligo-mediated GA=VI748/MAID6822 zygote oligo _VHΘ 1-69 MreI del zygotic oligo GTGAAAACCCACATCCTGAGAGTG
ACAAAAACCCTGAGGGAGAAGGCAGCTGTGCCGGGCTGAGGAGATGACAGGGGTTA
(SEQ ID NO: 30) Jxn PCR ΔMreI 5' detection V _H 1-69 FR3 ACAGAAGTTCCAGGGCAGAG
(SEQ ID NO: 31) 634 3' downstream detection M1116 (h70) CCCCCTCTTGCTCTCTTTCT
(SEQ ID NO: 32)

Example 2. Generation of Rodents Containing NP-Modified V _H Gene Segments

This example relates to a non-human animal whose genome comprises an immunoglobulin heavy chain variable region comprising a NP-modified _VH gene segment, eg, a _VH gene segment comprising the C-terminal tail of ANP (e.g., , rodents) to demonstrate production.

The VI748 targeting vector is linearized and is homozygous for the endogenous Ig heavy chain variable region locus containing deletions of all endogenous V _H , D _H , and J _H segments except for the most 5' V _H 1-86 segment (1115KO); Electroporation into mouse embryonic stem cells with a genome homozygous for the endogenous Ig light chain variable region κ locus, including replacement of all endogenous VK and JK segments with a complete repertoire of human VK and JK segments. The Ig heavy chain loci (50% Balb, 25% C57BL/6, 25% 129 background) of ES cells used for electroporation of each targeting vector are shown in FIG. 4 . After electroporation, the electroporated cells were cultured in selective medium. Drug-resistant colonies were picked 10 days after electroporation and as previously described (Valenzuela et al., supra ; Frendewey, D. et al., 2010, Methods Enzymol. 476:295-307, incorporated herein by reference). incorporated herein), screened by TAQMAN™ and karyotyping for accurate targeting using primer/probe sets that detect proper integration of the anchor modified V _H 1-69 gene segment.

Forward Primer: TGTGTCCTGTCCACAGGTG (SEQ ID NO: 34)

Probe: CCAGTCCAACAGTTCCGGTACG (SEQ ID NO: 35)

Reverse Primer: CAGCTGGACCTGGCTACC (SEQ ID NO: 36)

The VELOCIMOUSE® method (DeChiara, TM, et al., 2010, Methods Enzymol. 476:285-294; DeChiara, TM, 2009, Methods Mol. Biol. 530:311-324; Poueymirou et al., 2007, Nat. Biotechnol. 25:91- 99; incorporated herein by reference in its entirety), the targeted ES cells were injected into uncondensed 8-cell stage Swiss Webster embryos, heterozygous for an anchor (ANP) modified V _H segment and anchoring (ANP) Healthy, intact ES cell-derived F0 generation mice expressing the modified antibody were produced. Such modified mice are referred to herein as ANP-V _H 1-69 modified mice.

e.g., by subsequent addition of a recombinase (e.g., by Cre treatment) or in a Cre deleter mouse strain (to remove any lox selected cassette introduced by the targeting construct that was not deleted at the ES cell stage or the embryonic stage). The drug selection cassette can be selectively removed by crossing with, for example, WO 2009/114400). Optionally, the selection cassette can be maintained in mice.

Example 3. Immunophenotyping of rodents by flow cytometry

To determine the immunophenotype of ANP-V _H 1-69 transformed mice, bone marrow and spleen B cells were analyzed by flow cytometry. For this study, two VELOCIMMUNE® control mice (see, eg, US Pat. Nos. 8,502,018; 8,642,835; 8,697,940, each of which is incorporated herein by reference in its entirety) and Example 2 The three ANP-V _H 1-69 transformed mice described were sacrificed and their spleens and bone marrow harvested. Bone marrow was harvested from the femur by centrifugation at 8,000 rpm for 2 minutes. Spleens were dissociated to single cell suspensions. Red blood cells from spleen and bone marrow preparations were lysed with ACK lysis buffer and washed with DPBS containing 2% FBS. Isolated cells (1x10 ⁶ total) were incubated with anti-mouse CD16/CD32 (clone 2.4G2, BD) for 10 minutes on ice and then labeled with the antibody panel described in Table 2 for 30 minutes on ice.

Panel of Abs used for flow cytometry Bone Marrow Mature Ab Panel antigen clone sauce CD43 1B11 BioLegend c-kit 2B8 BioLegend IgM II/41 eBioscience IgD 11-26c.2a BioLegend B220 RA3-6B2 eBioscience CD19 ID3 BD CD3 17-A2 BioLegend Bone Marrow and Spleen Kappa/Lambda Panel antigen clone sauce IgK 187.1 BD IgL RML-42 BioLegend IgM II/41 eBioscience IgD 11-26c.2a BioLegend CD3 17A2 BioLegend B220 RA3-6B2 eBioscience CD19 ID3 BD Spleen Maturation Panel antigen clone sauce CD23 B3B4 BioLegend CD93 AA4.1 BioLegend IgM II/41 eBioscience IgD 11-26c.2a BioLegend CD19 ID3 BD CD21/35 7G6 BD B220 RA3-6B2 eBioscience

After staining, cells were washed and fixed with 2% formaldehyde. Data collection was performed on a BD LSRFortessa flow cytometer and analyzed with FlowJo software. Subsets of cells were identified using the following strategy. Bone marrow maturation: immature B cells (B220int IgM+), mature B cells (B220high IgM+), pro-B cells (IgM- B220int, c-KitintCD43high), pre-B cells (IgM- B220int, c-Kit-CD43int). Spleen and myeloid kappa/lambda: B cells (CD19+ CD3-), T cells (CD3+ CD19-), IgK+ B cells (CD19+ IgK+ IgL-), IgL+ B cells (CD19+ IgK- IgL+). Spleen maturation: mature B cells (CD19+, B220+ CD93-), follicular B cells (CD19+, B220+ CD93-, CD21/35int IgMint/+), marginal zone B cells (CD19+, B220+ CD93-, CD21/35+ IgM+) , metastatic B cells (CD19+, B220+ CD93+), T1 B cells (CD19+, B220+ CD93+, IgM+ CD23-), T2 B cells (CD19+, B220+ CD93+, IgM+ CD23+), and T3 B cells (CD19+, B220+ CD93+, IgMint CD23+). ).

As shown in Figures 5A-5F , in the spleen, there were similar levels of B cells in ANP-V _H 1-69 transformed mice compared to VELOCIMMUNE® control mice. The frequency of mature B cells in ANP-V _H 1-69 transformed mice appears to be similar to that observed in VELOCIMMUNE® control mice, but in immature cells compared to VELOCIMMUNE® control mice in ANP-V _H 1-69 transformed mice. There was a slight decrease. In the spleen, the frequencies of lambda and kappa positive B cells are similar in ANP-V _H 1-69 transformed mice compared to VELOCIMMUNE® control mice.

As shown in Figures 6A-6F , in the bone marrow, there were similar levels of B cells in ANP-V _H 1-69 transformed mice compared to VELOCIMMUNE® control mice. In bone marrow, there were more pro-B cells and fewer pre-B cells in ANP-V _H 1-69 transformed mice compared to VELOCIMMUNE® control mice. There were less mature and more immature B cells in the bone marrow of ANP-V _H 1-69 transformed mice compared to VELOCIMMUNE® control mice.

Additional immunophenotyping For ANP-V _H 1-69 transformed mice, levels of mouse IgG were analyzed via Western blot. For analysis, blood was drawn from a subset of ANP-V _H 1-69 modified mice and VELOCIMMUNE® control mice. Serum was collected in a serum separation tube (BD), incubated for 30 min, and separated from blood by centrifugation at 9000 rcf for 5 min at 4°C. Mouse serum was diluted 1:25 in PBS and then run on a 4-20% Novex Tris-glycine gel under non-reducing conditions. Gels were transferred to polyvinylidene difluoride (PVDF) membranes according to the manufacturer's specifications (BioRad Trans-Blot Transfer System). Blots were blocked overnight with 10% nonfat milk in Tris buffered saline containing 0.05% Tween-20 (TBST, Sigma). PVDF membranes were diluted 1:20,000 in 4% non-fat milk in TBST with anti-mouse IgG-HRP (Thermo/Pierce, Cat #31432) and incubated for 1 hour at room temperature. Blots were then washed 5 times for 5 minutes each wash and then developed for 1 minute with Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences) according to the manufacturer's specifications. Blots were then taken using a GE Healthcare ImageQuant LAS-4000 Cooled CCD Camera Gel Documentation System. Images were captured at 15 second intervals until either 20 images were captured or the images were fully exposed.

As shown in Figure 7 , serum IgG levels were comparable between ANP-V _H 1-69 modified mice and VELOCIMMUNE® control mice as measured by Western blot. These results suggest that ANP mouse Designer V mice have normal Ab levels compared to VELOCIMMUNE® control mice.

Additional immunophenotyping For ANP-V _H 1-69 transgenic mice, levels of total serum IgM and IgG were measured by ELISA. For ELISA, plates were coated with 1 μg/mL of anti-mouse IgM+IgG+IgA (Clone Ab102445, Abcam) overnight at 4°C. Plates were then washed in DPBS with 0.1% Tween-20 and blocked in 1% BSA in DPBS for 1 hour at room temperature. Serum for ANP-V _H 1-69 modified mouse or VELOCIMMUNE® control mouse and mouse IgM standard (Biolegend, Cat# 401604) or mouse IgG standard (Sigma, Cat# I8765) were serially diluted in 1% BSA in DPBS and Incubated for 1 hour at room temperature. After incubation, plates were washed in DPBS with 0.1% Tween-20 and incubated with anti-mouse IgM-HRP (Southern Biotech, Cat #1021-05) or anti-mouse IgG-HRP (Southern Biotech, Cat #1030-05). were used to detect IgM or IgG. After an additional wash was performed, TMB substrate (BD) was added. After allowing color development, the reaction was stopped with 1N sulfuric acid and absorbance was read at 450 nm on a SpectraMax plate reader. Standard plots were generated in GraphPad Prism using non-linear regression of IgM or IgG standards (curved fit, 4 parameters) and serum IgM and IgG levels were quantified.

As shown in Figures 8A-8B , serum IgG levels measured by ELISA were comparable between ANP-V _H 1-69 modified mice and VELOCIMMUNE® control mice. Serum IgM levels measured by ELISA were also comparable between ANP-V _H 1-69 modified mice and VELOCIMMUNE® control mice. Both results suggest that ANP mice have normal Ab levels compared to VELOCIMMUNE® control mice.

Retention of the anchor by the antibody was confirmed by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF MS). Immunoglobulins isolated from mouse serum using Protein A magnetic beads (Thermo Scientific) were run by SDS-PAGE gel under reducing conditions to separate immunoglobulin heavy and light chains. Immunoglobulin heavy chains were excised and digested overnight with Lys-C enzyme (Promega). Salt was removed from the digestion with a C18 Ziptip (Millipore) according to the manufacturer's protocol. Peptides were eluted from each ziptip in 2.5 ul of 70% ACN/0.1% TFA containing 10 mg/ml a-cyano-4-hydroxycinnamic acid (CHCA), 70% acetonitrile +0.1% It was applied directly to a Bruker Anchorchip Target (Bruker Daltonics) mixed with a-cyano-4-hydroxycinnamic acid (Protea), dissolved in trifluoroacetic acid (TFA). Upon drying, each target was analyzed in reflection-positive mode on a Bruker Ultraflextreme MALDI MS (Bruker Daltonics) by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF MS).

MALDI-TOF MS suggests that ANP-modified immunoglobulins remain intact in serum of ANP-V _H- 1-69 modified mice (data not shown).

Example 4. Production of Antibodies in Rodents Containing Engineered Immunoglobulin Variable Region Gene Segments

This example shows antibody production in a rodent whose genome comprises an immunoglobulin heavy chain variable region comprising an immunoglobulin variable region gene segment engineered as described herein. The methods described in this Example, and/or immunization methods well known in the art, can be performed using a polypeptide or fragment thereof (e.g., a peptide derived from a desired epitope), or a combination of desired polypeptides or fragments thereof, as described herein. It can be used to immunize rodents containing immunoglobulin variable region gene segments such as

In summary, a cohort of mice comprising an engineered immunoglobulin variable region gene segment as described herein is subjected to an antigen of interest that binds to an antigen of interest, e.g., a cognate receptor, using immunization methods known in the art. Recipients of non-immunoglobulin polypeptides were inoculated. Antibody immuno-responses were monitored by ELISA immunoassay (ie serum titers).

immunization

Using standard immunization protocols, VELOCIMMUNE® controls (n=3) and ANP-V _H 1- 69 modified (n=4) mice were immunized. Mice were bled before initiation of immunization and after immunogen boost. Human NPR3 protein (consisting of the extracellular domain of NPR3 fused to a myc-myc-hexahistidine tag; also referred to as human NPR3 ecto-MMH) and engineered human NPR3 expression for the last bleed before mice were euthanized for antibody isolation. A titer assay was performed on cells (HEK293 cells engineered to overexpress full-length human NPR3; also referred to as 293/hNPR3 cells).

Determination of anti-serum titer

Antibody titers in serum against NPR3 were determined using ELISA. 96-well microtiter plates (Pierce) were coated with 2 μg/mL of human NPR3 ecto-MMH antigen in phosphate buffered saline (PBS, Irvine Scientific) overnight at 4°C. Plates were washed with phosphate buffered saline (PBS-T, Sigma-Aldrich) containing 0.05% Tween-20 and blocked with 250 μl 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 hour at room temperature. . Pre-immune and immunized anti-serum were serially diluted 3-fold in 0.5% BSA-PBS and added to the plate for 1 hour at room temperature. Plates were washed and goat anti-mouse IgG-Fc-horseradish peroxidase (HRP) conjugated secondary antibody (Jackson Immunoresearch) was added to the plate at a 1:5000 dilution and incubated for 1 hour at room temperature. Plates were washed and developed by incubation for 15-20 minutes using TMB/H2O2 as substrate. The reaction was quenched with acid and the plate was read on a spectrophotometer (Victor, Perkin Elmer) at 450 nm. Antibody titers were calculated using Graphpad PRISM software. The titer was defined as the interpolated serum dilution factor at which the binding signal was twice the background.

Antibody titer in cells

Anti-NPR3 antibody titers in engineered cells were determined using the Meso Scale Discovery (MSD) MULTI-ARRAY® technology. A 96-well carbon electrode plate (available from MSD) was coated with 40,000 cells per well of 293/hNPR3 cells or HEK293 cells in PBS for 1 hour at 37°C. The cell coating solution was decanted and blocked by incubation with 150 μL of 2% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 hour at room temperature (RT), followed by washing with PBS. Pre-immune and immunized anti-serum were serially diluted 3-fold in 1% BSA-PBS and added to the plate for 1 hour at room temperature followed by washing. Goat anti-mouse IgG-Fc ruthenium conjugated secondary antibody (Jackson ImmunoResearch and Ruthenium labeled in-house) was then added to the plate at 1 μg/mL and incubated for 1 hour at room temperature. MSD's 4X Read Buffer T surfactant-free was diluted 1X and 150 μL was added to each well and read on the MSD SECTOR® imager instrument. Antibody titers were calculated using Graphpad PRISM software. The titer was defined as the interpolated serum dilution factor at which the binding signal was twice the background.

result

After immunization with the NPR3 ecto protein immunogen, the humoral immune-response in VELOCIMMUNE® control and ANP-V _H 1-69 transformed mice was determined using recombinant hNPR3 protein and engineered human NPR3-expressing cells (HEK293/hNPR3). decided. Antisera from ANP-V _H 1-69 modified mice (n=4) had an average titer of 465,625, comparable to an average titer of 394,032 for the VELOCIMMUNE® control strain (n=3), on the hNPR3.mmh protein. A range of high antibody titers were shown ( FIG. 9 ). The average antibody titers of ANP-V _H 1-69 transformed mice in HEK293/hNPR3 expressing cells and parental HEK293 cells were 268,763 and 3,948, respectively, suggesting that the anti-serum was specific for NPR3. Similar results were obtained in VELOCIMMUNE® control mice, with average antibody titers of 72,826 and 5,219 in HEK293/hNPR3 expressing cells and parental HEK293 cells, respectively ( FIG. 10 ). These results suggest that ANP-V _H 1-69 modified mice are capable of receiving an immune-response similar to the VELOCIMMUNE® control mouse strain.

Example 4. Isolation of Cells Encoding and/or Expressing Antibodies Produced in Rodents Containing Engineered Immunoglobulin Variable Region Gene Segments

When the desired immune response is achieved, splenocytes (and/or lymphoid tissue) are harvested and fused with mouse myeloma cells to preserve viability and form an immortal hybridoma cell line. The hybridoma cell lines are screened (eg, by ELISA assay) and selected to identify hybridoma cell lines that produce antigen-specific antibodies. Hybridomas can be further characterized for the desired relative binding affinity and isotype. Using this technique, several antigen-specific chimeric antibodies (ie, antibodies comprising human variable domains and rodent constant domains) are obtained.

DNA encoding the heavy and light chain variable regions can be isolated and ligated to the desired isotypes (constant regions) of the heavy and light chains for the production of fully human antibodies. Such antibody proteins can be produced in cells such as CHO cells. The fully human antibody is then characterized for the relative binding affinity and/or neutralizing activity of the antigen of interest.

DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes. Initially, high affinity chimeric antibodies having human variable regions and rodent constant regions are isolated, characterized and selected for desired properties including affinity, selectivity, epitope, etc. Rodent constant regions are substituted with desired human constant regions to generate fully human antibodies. Although the constant region selected can vary widely depending on the specific application, the high affinity antigen binding properties and target specificity properties reside in the variable region. Antigen-specific antibodies may also be generated from antigen-positive B cells (derived from immunized mice) without fusion of myeloma cells, e.g., as specifically described in U.S. Patent No. 7,582,298, the contents of which are specifically incorporated herein by reference. are isolated directly Using this method, several fully human antigen-specific antibodies (ie, human variable domains and human constant domains) are prepared.

Specifically, splenocytes from NPR3 immunized VELOCIMMUNE® mice or ANP-V _H 1-69 transformed mice were cultured with a C-terminal human Fc tag (hNPR3 ecto-hFc) and US Pat. No. 7,582,298 (see in its entirety). Incorporated herein as), human NPR3 extracellular domain expressed with FITC-anti-mFc was stained. Single IgG positive and antigen positive B cells were isolated into separate wells of a 384-well plate by fluorescence-activated cell sorting (FACS). RT-PCR of antibody genes from these B cells was performed according to the method described by Wang and Stollar (Journal of Immunological Methods 2000; 244: 217-225, incorporated herein by reference in its entirety). Briefly, cDNA for each B cell was synthesized via a reverse transcriptase (RT) reaction (SUPERSCIPT ^™ III, Invitrogen). Each resulting RT product was then split and transferred to two corresponding wells on a separate 384-well plate for amplification of heavy and light chain sequences. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for the human IgG heavy chain variable region leader sequence and a 3' primer specific for the mouse heavy chain constant region to form amplicons. A 5' degenerate primer set specific for framework 1 of human IgG heavy chain variable region sequence or a primer set specific for ANP peptide (ANP mouse only) and a 3' degenerate primer specific for framework 4 of human IgG heavy chain variable region sequence. Using the set, amplicons were applied in a second round. Another set of resulting RT products was first amplified by PCR using a set of 5' degenerate primers specific for the human kappa light chain variable region leader sequence and a 3' primer specific for the mouse kappa light chain constant region to form amplicons. A second round of PCR was performed on this amplicand using a 5' degenerate primer set specific for framework 1 of the human kappa light chain variable region sequence and a 3' degenerate primer set specific for framework 4 of the human kappa light chain variable region sequence. performed. Heavy and light chain PCR products were cloned into antibody vectors containing an IgG heavy chain constant region and a kappa light chain constant region, respectively. Recombinant hIgG1 antibodies were produced by transient transfection of CHO K1 cells for further screening.

Example 5: Primary screening of supernatants isolated from CHO K1 cells

Binding properties of a panel of anti-NPR3 monoclonal antibodies containing supernatants from CHO K1 transfected cells, 146 supernatants from ANP-V _H 1-69 transfected mice and 65 supernatants from VELOCIMMUNE® controls were determined. were characterized, and kinetic binding parameters and equilibrium binding constants were determined using SPR techniques.

Equilibrium dissociation constants (K _D ) and dissociation rates at pH6.0 for NPR3 binding to different NPR3 monoclonal antibodies (mAbs) were determined using real-time surface plasmon resonance using a Biacore 4000 instrument. All binding studies were performed in 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl and 0.05% v/v surfactant Tween-20 (PBS-T) prepared in pH 7.4 (PBS-T_pH7.4) running buffer at 25 °C. . The Biacore CM5 sensor chip surface was first derivatized by amine linkage with an anti-human Fc specific mouse mAb (REGN2567) to capture different NPR3 mAbs. A single 100 nM concentration of the ectodomain of human NPR3 expressed with a C-terminal myc-myc-6xHis tag (hNPR3-MMH) prepared in PBS-T_pH7.4 running buffer was applied over the NPR3 mAb capture surface at a flow rate of 30 μL/min. Inject for 90 seconds and dissociation in PBS-T_pH7.4 running buffer was monitored for 90 seconds. After the dissociation step, PBS-T buffer prepared at pH6.0 (PBS-T_pH6.0) was injected for 2 minutes. At the end of each cycle, the NPR3 mAb capture surface was regenerated using a 12 second injection of 20 mM phosphoric acid.

Association rates ( ka ₎ and dissociation rates ( k _d ) were determined by fitting real-time binding sensorgrams to a 1:1 binding model with mass transport constraints using Biacore 4000 evaluation software and NPR3 mAb in PBS-T_pH6.0. Dissociation of hNPR3-MMH from was determined by fitting dissociation curves using Scrubber 2.0c curve-fitting software. The association dissociation equilibrium constant (K _D ) and dissociation half-life (t½) were calculated from the kinetic rates as follows:

Median and mean values of K _D and t½ values of NPR3 mAb isolated from VELOCIMMUNE® control and ANP-V _H 1-69 transgenic mice were also calculated and provided in Table 3.

The K _D and t½ values of NPR3 mAb isolated from VELOCIMMUNE® control and ANP-V _H 1-69 transgenic mice were compared using box and whisker plots as shown in FIGS. 11-12 , respectively.

The average and median values of K _D and t _1/2 of the ANP-V _H 1-69 modified and VELOCIMMUNE® control mAb groups are listed in Table 3, respectively, and the comparison of K _D values and t _1/2 is shown in the figure, respectively. 11 and 12 are shown. Both the ANP-V _H 1-69 modified and VELOCIMMUNE® control mice were able to produce antibodies in a wide affinity range, and the _KD values for the Abs of ANP-V _H 1-69 modified mice were higher than those of the VELOCIMMUNE® control mice. The mean/median of was smaller, and t _1/2 was longer. These results suggest that ANP-V _H 1-69 modified mice are capable of producing high affinity antibodies in a higher population than VELOCIMMUNE® control mice.

Mean and median K D and _t½ values of hNPR3-MMH binding to NPR3 monoclonal antibodies (mAbs) isolated from VELOCIMMUNE® control and ANP-V _H 1-69 transformed mice at 25°C. mouse strain number of samples Central KD (M) Mean KD (M) central t½
(minute) Average t½
(minute) ANP-V _H 1-69 modified 146 2.18E-09 7.85E-09 47.00 48.64 VELOCIMMUNE® Control 65 6.05E-09 1.39E-08 6.00 27.36

Luminex

A Luminex binding assay was performed to determine the binding of antibodies isolated from mice immunized with NPR3 to a panel of NPR3 antigens. For this assay, antigens were amine conjugated to Luminex microspheres. Microspheres for amine binding proteins were prepared as follows: Approximately 10 million MicroPlex microspheres (MicroPLex Microspheres, Luminex, Cat. No. LC10000-00) were placed in 500 μL of 0.1 M NaPO4, pH 6.2 (activation buffer) on a vortexer. After resuspension, the supernatant was removed by centrifugation. The microspheres were resuspended in 160 L of activation buffer, 15 L of 50 mg/mL N-hydroxysuccinimide (NHS, Thermo Scientific, Cat# 24525) was added at 25°C, followed by the addition of 50 mg/mL of 15 L of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, ThermoScientific, Cat# 22980) was added to activate the carboxylic acid group (-COOH). After 10 minutes, the pH of the reaction was reduced to 5.0 by adding 500 μL of 50 mM MES pH 5.0, the microspheres were vortexed, and the supernatant was removed by centrifugation. Activated microspheres were mixed with 25 μg/mL of protein antigen [human NPR3 extracellular domain expressed with a C-terminal human Fc tag (hNPR3 ecto-hFc) and C-terminal complexed with human ANP in 50 mM MES pH 5.0]. human NPR3 extracellular domain expressed with a mouse Fc tag (hNPR3-ecto-mFc-hANP)] and immediately mixed with 500 μL. A unique bead region per bound protein/antibody was used. The microsphere-protein mixture was incubated at 25° C. for 2 hours. The binding reaction was quenched by the addition of 50 μL of 1 M Tris-HCl, pH 8.0, and the microspheres were vortexed, centrifuged, and washed three times with 800 μL of PBS containing 0.05% Tween20 to remove unbound protein and Other reaction components were removed. Microspheres were resuspended in PBS at 10 million microspheres/mL with 2% BSA and 0.05% Na azide.

Microspheres with amine-binding proteins were mixed at 2700 beads/mL, then 75 μL of microspheres were plated per well in 96 well filter plate flat bottom plates (Millipore, Cat. No: MSBVN1250) and individual anti- It was mixed with 25 μL of supernatant containing NPR3 antibody. Samples and microspheres were incubated at 25° C. for 2 hours and then washed twice with 200 μL of PBS with 0.05% Tween 20. To detect the level of antibody bound to individual microspheres, 2.5 μg/mL R-phycoerythrin conjugated goat F(ab')2 anti-human kappa (Southern Biotech , Cat. No. 2063-09) was added, followed by incubation at 25° C. for 30 minutes. Samples were washed twice with 200 μL of PBS containing 0.05% Tween 20 and resuspended in 150 μL of PBS containing 0.05% Tween 20. Plates were read on a Luminex FlexMap 3D instrument using Luminex xPonent software version 4.2.

Antibodies that bound to hNPR3-ecto-hFc and hNPR3-ecto-mFc-hANP with binding signals greater than 1000, less than 5000 and greater than 5000 are shown in Table 4. A total of 439 samples were tested: 236 samples from ANP-V _H 1-69 modified mice and 203 samples from VELOCIMMUNE® control mice. The binding signal was measured as the median of fluorescence intensity (MFI). As shown in Table 4, ANP-V _H 1-69 modified mice bound to complexes of hNPR3 ecto-hFc and hNPR3 ecto-mFc-hANP >5000 in the Luminex assay, compared to antibodies isolated from VELOCIMMUNE® control mice. The percentage of antibodies from was higher.

Total number (percentage) of anti-NRP3 antibodies bound to amine conjugated hNPR3-ecto-hFc and hNPR3-ecto-mFc-hANP in Luminex binding assay. mouse strain Combined signal (MFI) hNPR3-ecto-hFc hNPR3-ecto-mFc-hANP ANP- V _H 1-69
transformed >5000 206 (87%) 202 (86%) >1000 14 (6%) 18 (8%) VELOCIMMUNE®
control group >5000 88 (43%) 83 (41%) >1000 14 (22%) 50 (25%)

blocking function

An ELISA-based blocking assay was developed to determine the ability of anti-NPR3 antibodies to block human NPR3 binding human ANP (hANP). In this assay, a recombinant human comprising a portion of the human NPR3 extracellular domain (amino acids Gly27-Ser482) fused to the Fc portion of mouse IgG2a at the mouse IgG2a at the NPR3 C-terminus (hNPR3-mFc, Seq ID or accession number). NPR3 protein was passively absorbed overnight at 4° C. at a concentration of 2 μg/mL in PBS on 96-well microtiter plates. Subsequently, non-specific binding sites were blocked using a 0.5% (w/v) BSA solution in PBS for 1 hour at room temperature. After washing the plate with PBS+ tween, a 1:10 dilution of anti-NPR3 antibody supernatant or 1 μg/mL of non-binding human IgG1 isotype control antibody was added. Plates were incubated at room temperature for 1 hour, then recombinant biotinylated hANP (biotin-hANP; Phoenix Pharmaceuticals INC, Buringame, Calif.) was added to a final concentration of 200 pM and incubated for 1 hour. The concentration of biotin-hANP was in the dynamic range of dose dependent binding of biotin-hANP to plate-coated hNPR3 around the EC50 value. Plates were washed, plate-bound biotin-hANP was detected with 100 ng/mL streptavidin conjugated with horseradish peroxidase (HRP), and TMB substrate solution (BD Biosciences, Calif. San Jose material) was used to visualize. Absorbance at 450 nm was measured on a Victor ^™ multi-label plate reader (PerkinElmer ^™ ).

Percent blocking for the tested anti-NPR3 antibodies was calculated using the formula:

Antibodies that blocked the binding of biotin-hANP to hNPR3 by 50% or more were classified as blocking agents.

The ability of anti-NPR3 antibody supernatants from VELOCIMMUNE® control and ANP-V _H 1-69 modified mice to block human ANP binding to human anti-NPR3 was evaluated using an ELISA-based blocking assay. As shown in Table 5, 338 anti-NPR3 antibody supernatants were tested to block human ANP binding to human NPR3, of which 125 were from VELOCIMMUNE® control mice and 213 were from ANP-V _H 1-69 It was derived from a modified mouse. Supernatants of 134 anti-NPR3 antibodies blocked hNPR3-mFc binding to biotin-hANP with a blocking percentage greater than 50%. Of the 134 blockers, 17 were isolated from VELOCIMMUNE® control mice and 117 were isolated from ANP-V _H 1-69 transformed mice, which were from VELOCIMMUNE® control mice and ANP-V _H 1-69 transformed mice. 14% and 55%, respectively, of the number of antibodies tested. Of the top 50 blockers, 7 were from VELOCIMMUNE® control mice and 43 were from ANP-V _H 1-69 transformed mice, representing 6% and 20% of the total mAbs tested from the two strains of mice, respectively. occupies

In this experiment, the human IgG1 isotype control antibody showed a blocking percentage of 32% and was classified as a non-blocker.

Overall, compared to VELOCIMMUNE® control mice, ANP-V _H 1-69 modified mice produced more antibodies that bind NPR3 with high specificity and block recombinant NPR3 protein binding to human ANP.

Summary of supernatants of anti-NPR3 antibodies blocking human ANP binding to human NPR3-mFc. mouse strain Number of anti-NPR3 tested number of blockers
(% Blocking >50%) per mouse strain
blocker % Number of mAbs among the top 50 blockers
(Range of Blocked %) % blockers with highest 50 values for % block from individual strains of mice VELOCIMMUNE®
control group 125 17 14% 7
(80% - 95%) 6% ANP- _VH 1-69
transformed 213 117 55% 43
(80% - 99%) 20%

The ability of anti-human NPR3 monoclonal antibodies isolated from ANP-V _H 1-69 modified mice and VELOCIMMUNE® control mice immunized with NPR3 recombinant protein to bind to human NPR3 (hNPR3) expressing cells was measured by electrochemiluminescence (ECL). It was determined using based analysis.

Briefly, HEK293 was engineered to express human NPR3 by transfecting cells with a neomycin-resistant pLVXN.NPR3 expression plasmid encoding human NPR3 (amino acids M1-A541, UniProtKB - P17342). Non-transfected HEK293 cells that did not show detectable expression of NPR3 by fluorescence activated cell sorting (FACS) with commercial atrial natriuretic peptide (ANP) were included in the experiment as a background binding control. An anti-allergen human IgG1 antibody and an anti-idiotypic mouse IgG2 antibody were included as irrelevant antibody controls for binding.

Experiments were conducted according to the following procedure. NPR3-expressing cells and parental cells cultured in flasks were rinsed once in Ca2+/Mg2+-free 1xPBS buffer, incubated with enzyme-free cell dissociation solution at 37°C for 10 minutes, and then cells were detached from flasks. All cells were washed once with 1xPBS containing Ca2+/Mg2+ and counted with a Cellometer™ Auto T4 cell counter (Nexcelom Bioscience LLC, Lawrence, MA). Approximately 2.0×10 4 HEK293/hNPR3 or HEK293 cells per well were seeded separately on 96-well carbon electrode plates (Meso Scale Discovery, Rockville, MD) and incubated for 1 hour at 37° C. to allow the cells to adhere. Non-specific binding sites were blocked by incubation with 2% BSA (w/v) in 1xPBS containing Ca2+/Mg2+ for 1 hour at room temperature. To plate-bound cells, anti-NPR3 antibody supernatant was added at either 1:10 or 0.2 μg/mL control antibody dilution and then incubated for 1 hour at room temperature. The plate was then washed to remove unbound antibody using an AquaMax2000 plate washer equipped with a cell wash head (MDS Analytical Technologies, Sunnyvale, Calif.). Plate-bound human IgG was detected with SULFO-TAG™-conjugated goat polyclonal anti-human IgG antibodies (Jackson Immunoresearch Labs, West Grove, Pa.) specific for heavy and light chains, and plate-bound mouse IgG ( commercial NPR3 antibody and mIgG isotype control antibody) at room temperature for 1 hour using a SULFO-TAGTM-conjugated goat polyclonal anti-mouse IgG antibody (Jackson Immunoresearch Labs, West Grove, PA) specific for the Fcγ fragment. detected. After washing, the plate was developed with read buffer (Meso Scale Discovery, Rockville, MD) according to the manufacturer's recommended procedure, and the luminescence signal was recorded with a SECTOR Imager 600 (Meso Scale Discovery, Rockville, MD). The binding signal ratio of HEK293/hNPR3 to parental HEK293 cells was calculated and reported as an indication of the binding specificity of the anti-NPR3 antibody. Antibodies with a binding signal to HEK293/hNPR3 cells of 200 RLU or more and binding ratios of 3 or more were classified as specific binding agents, and antibodies with binding signals of less than 200 RLU or binding ratios of less than 3 were classified as nonspecific binding agents or non-binding agents. classified as characters.

result:

The ability of anti-NPR3 antibodies isolated from VELOCIMMUNE® control and ANP-V _H 1-69 transformed mice to specifically bind to NPR3-expressing HEK293 cells was evaluated in an electrochemiluminescence-based binding assay.

The experimental results are summarized in Table 6. A total of 338 anti-NPR3 antibodies in crude supernatant were tested for binding to HEK293/hNPR3 cells, of which 125 antibodies were isolated from VELOCIMMUNE® control mice and 213 antibodies were ANP-V _H 1-69 modified. isolated from mice. A total of 250 anti-NPR3 antibodies, 62 from the VELOCIMMUNE® control and 188 from ANP-V _H 1-69 transformed mice, had a binding signal of >200 RLU in HEK293/hNPR3 cells and a binding signal of >3 in HEK293/hNPR3 to parental HEK293. Binding ratios are shown, indicating these antibodies can specifically bind to NPR3. 50% of anti-NPR3 antibodies in VELOCIMMUNE® control mice compared to 88% of ANP-V _H 1-69 modified mice were specific NPR3 binders. Of the top 50 specific binders, 6 antibodies were from VELOCIMMUNE® control mice and 44 antibodies were from ANP-V _H 1-69 transgenic mice, which were 5 of the anti-NPR3 antibody supernatants from VELOCIMMUNE® control mice. % and 21% of anti-NPR3 antibody supernatants from ANP-V _H 1-69 modified mice, suggesting that they are the top binders.

In the experiment, both the human IgG1 and mouse IgG2 isotype controls, the commercial anti-hNPR3-mIgG2 antibody included as a positive control that specifically bound to HEK293/hNPR3 cells, did not bind HEK293/hNPR3 cells as expected.

Overall, the binding results using HEK293/hNPR3 cells showed that ANP-V _H 1-69 transformed mice could produce antibodies that specifically bound to hNPR3-expressing cells, and that the high potency of the binding agent enriched ANP-V _H Antibodies isolated from 1-69 modified mice may be higher than normal VELOCIMMUNE® control mice.

Summary of supernatants of anti-NPR3 antibodies binding to cells engineered to express human NPR3. mouse strain Number of anti-NPR3 tested Number of specific anti-NPR3 binders % of specific anti-NPR3 binders per mouse strain Number of specific anti-NPR3 binders with top 50 cell binding ratios % of anti-NPR3 specific binders with the top 50 cell binding ratios per total binders per strain of mice VELOCIMMUNE®
control group 125 62 50% 6 5% ANP- _VH 1-69
modified mouse 213 188 88% 44 21%

purified antibody

To evaluate the ability of anti-NPR3 antibodies derived from ANP-V _H 1-69 transformed mice to bind NPR3, engineered cell lines were transfected to stably express full-length human NPR3 in HEK293 cells (human embryonic kidney 293, ATCC) and then sorted for high expression. The resulting cell line was named HEK293/hNPR3 high class (ACL#9752, Regeneron).

For flow cytometric binding assessment, HEK293/hNPR3 highly sorted cells were lifted into enzyme-free cell dissociation buffer (Millipore, cat#S-004-C), washed once and stained in staining buffer (1X PBS, without calcium or magnesium (Irvine). Scientific, cat#9240) containing 2% filtered fetal bovine serum (Seradigm, cat#1500-500)), stained with fixable green visualization dye (Life Technologies, cat#L23101), washed again and V- Plated in a bottom staining plate (Axygen Scientific, cat#P-96-450-V-C-S).

Purified antibodies, commercial antibodies (GeneTex, GTX84015), or isotype control antibodies isolated from ANP-V _H 1-69 modified mice and VELOCIMMUNE® control mice were serially diluted 1:3 in staining buffer from 100 nM to 24.4 pM (with an additional well for staining buffer only, no test molecules) were added to the cells in the staining plate.

After an additional washing step, prior to fixation with CytoFix (BD Biosciences, cat#554655) fixation buffer, Alexa Fluor®647-tagged secondary detection antibody [anti-human (Jackson ImmunoResearch, cat#109-607-003) and anti- mice (Jackson ImmunoResearch, cat#115-607-003)]. All samples were filtered through a 96-well filter plate (Pall, cat#8027) into a U-bottom reading plate (Corning, cat#3799) and then run through an iQue PLUS flow cytometer and for each sample the mean fluorescence intensity ( MFI) was measured. Data were gated in iQue Forecyt® software to calculate the geometric mean of each sample in the RL1-A channel, and the results were further analyzed using a nonlinear regression (4-parameter logistic) model in Prism® 8 software to obtain EC50 values. The maximum fold combination was calculated using the formula:

In this equation, “MFI _{geometric mean RL1-A} ” refers to the maximum mean fluorescence intensity (MFI) value in the RL1-A channel from cells stained with the purified antibody. “MFI _{geometric mean RL1-A, secondary antibody only control} ” refers to the MFI value in the RL1-A channel from cells stained with Alexa647-tagged secondary detection antibody only.

As shown in Table 7, 12 purified anti-NPR3 antibodies (“+ANP”) isolated from ANP-V _H 1-69 modified mice expressed with an ANP tag, ANP-V _H expressed without an ANP tag, Specificity of HEK293/hNPR3 highly sorted cells for 12 purified anti-NPR3 antibodies (“-ANP”) isolated from 1-69 modified mice, and 8 purified anti-NPR3 antibodies isolated from VELOCIMMUNE® control mice. Enemy binding was tested by flow cytometry. 11 of 12 purified anti-NPR3 antibodies isolated from ANP-V _H 1-69 transgenic mice expressed with an ANP tag showed specific binding to HEK293/hNPR3 cells and a secondary detection antibody alone control Fold binding values for 21 to 1012 and EC50 values were 7.7 nM to >10 nM. Ten of 12 purified anti-NPR3 antibodies isolated from ANP-V _H 1-69 transformed mice expressed without an ANP tag showed specific binding to HEK293/hNPR3 cells, and a secondary detection antibody alone control Fold binding values ranged from 132 to 819 and EC50 values ranged from 6.3 nM to >10 nM. All eight purified anti-NPR3 antibodies isolated from VELOCIMMUNE® control mice showed specific binding to HEK293/hNPR3 cells, with fold binding values of 215 to 789 and 2.4 nM to >10 versus the secondary detection antibody only control. had an EC50 value of nM. The isotype control antibody did not bind HEK293/hNPR3 cells, and the commercial anti-NPR3 antibody bound HEK293/hNPR3 with 22-fold binding compared to the secondary detection antibody alone control and an EC50 of 7.6 nM.

antibody combined multiple EC ₅₀ [M] VELOCIMMUNE® Control Ab 1 609 >1.0E-08 VELOCIMMUNE® Control Ab 2 311 >1.0E-08 VELOCIMMUNE® Control Ab 3 215 2.4E-09 VELOCIMMUNE® Control Ab 4 562 1.1E-08 VELOCIMMUNE® Control Ab 5 629 1.0E-08 VELOCIMMUNE® Control Ab 6 789 >1.0E-08 VELOCIMMUNE® Control Ab 7 443 1.1E-08 VELOCIMMUNE® Control Ab 8 291 2.1E-08 ANP-V _H 1-69 Modified Ab 1-ANP 132 >1.0E-08 ANP-V _H 1-69 Modified Ab 2-ANP 798 >1.0E-08 ANP-V _H 1-69 Modified Ab 3-ANP 689 >1.0E-08 ANP-V _H 1-69 Modified Ab 4-ANP 798 7.0E-09 ANP-V _H 1-69 Modified Ab 5 -ANP 3 no bonding ANP-V _H 1-69 Modified Ab 6 -ANP 576 9.1E-09 ANP-V _H 1-69 Modified Ab 7 -ANP 605 7.0E-09 ANP-V _H 1-69 Modified Ab 8 -ANP 819 9.9E-09 ANP-V _H 1-69 Modified Ab 9 -ANP 566 6.3E-09 ANP-V _H 1-69 Modified Ab 10 -ANP 799 8.7E-09 ANP-V _H 1-69 Modified Ab 11 -ANP 712 1.7E-08 ANP-V _H 1-69 Modified Ab 12 -ANP 14 no bonding ANP-V _H 1-69 Modified Ab 1+ANP 330 >5.0E-08 ANP-V _H 1-69 Modified Ab 2+ANP 519 7.7E-09 ANP-V _H 1-69 Modified Ab 3+ANP 664 1.2E-08 ANP-V _H 1-69 Modified Ab 4+ANP 688 8.4E-09 ANP-V _H 1-69 Modified Ab 5+ANP 7 no bonding ANP-V _H 1-69 Modified Ab 6+ANP 549 1.0E-08 ANP-V _H 1-69 Modified Ab 7+ANP 686 1.3E-08 ANP-V _H 1-69 Modified Ab 8+ANP 783 9.5E-09 ANP-V _H 1-69 Modified Ab 9+ANP 913 1.1E-08 ANP-V _H 1-69 Modified Ab 10+ANP 1012 >1.0E-08 ANP-V _H 1-69 Modified Ab 11+ANP 411 1.5E-08 ANP-V _H 1-69 Modified Ab 12+ANP 21 >5.0E-08 Isotype Control Ab 1 2 no bonding Isotype Control Ab 2 One no bonding Commercial anti-hNPR3 Abs 22 7.6E-09

equivalent

Having described several aspects of at least one embodiment of the present invention, it will be appreciated by those skilled in the art that various changes, modifications and improvements will readily occur to those skilled in the art. These changes, modifications and improvements are intended to become part of this disclosure and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are exemplary only, and the invention is described in detail by the claims that follow.

Those skilled in the art will understand the usual errors or standard deviations that can be attributed to values obtained in the assays or other procedures described herein.

Publications, websites, and other reference materials are cited to explain the background of the present invention and to provide additional detail in view of its implementation being incorporated herein by reference.

SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. Mastaitis, Jason Murphy, Andrew J. McWhirter, John Voronina, Vera Gromada, Jesper <120> NUCLEIC ACIDS ENCODING ANCHOR MODIFIED ANTIBODIES AND USES THEREOF <130> 009108.507WO1/10507WO01 <150> 63/129,893 <151> 2020- 12-23 <150> 63/219,402 <151> 2021-07-08 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 855 <212> DNA <213> Homo sapiens <400> 1 gagacaggga cagacgtagg ccaagagagg ggaaccagag aggaaccaga ggggagagac 60 agagcagcaa gcagtggatt gctccttgac gacgccagca tgagctcctt ctccaccacc 120 accgtgagct tcctcctttt actggcattc cagctcctag gtcagaccag agctaatccc 180 atgtacaatg ccgtgtccaa cgcagacctg atggatttca agaatttg ct ggaccatttg 240 gaagaaaaga tgcctttaga agatgaggtc gtgcccccac aagtgctcag tgagccgaat 300 gaagaagcgg gggctgctct cagccccctc cctgaggtgc ctccctggac cggggaagtc 360 agcccagccc agagagatgg aggtgccctc gggcgggg cc cctgggactc ctctgatcga 420 tctgccctcc taaaaagcaa gctgagggcg ctgctcactg cccctcggag cctgcggaga 480 tccagctgct tcgggggcag gatggacagg attggagccc agagcggact gggctgtaac 540 agcttccggt actgaagata acagccaggg aggacaagca gggctgggcc tagggacaga 600 ctgcaagagg ctcctgtccc ctggggtctc tgctgcattt gtgtcatctt gttgccatgg 660 agttgtgatc atcccatcta agctgcagct tcctgtcaac acttctcaca tcttatgcta 720 actgtagata aagtggtttg atggtgactt cctcgcctct cccaccccat gcattaaatt 780 ttaaggtaga acctcacctg ttactgaaag tggtttgaaa gtgaataaac ttcag cacca 840 tggacagaag acaaa 855 <210> 2 <211> 151 <212> PRT <213> Homo sapiens <400> 2 Met Ser Ser Phe Ser Thr Thr Thr Val Ser Phe Leu Leu Leu Leu Leu Ala 1 5 10 15 Phe Gln Leu Leu Gly Gln Thr Arg Ala Asn Pro Met Tyr Asn Ala Val 20 25 30 Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu 35 40 45 Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser 50 55 60 Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val 65 70 75 80 Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala 85 90 95 Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys 100 105 110 Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser 115 120 125 Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu 130 135 140 Gly Cys Asn Ser Phe Arg Tyr 145 150 < 210> 3 <211> 5 <212> PRT <213> Homo sapiens <400> 3 Asn Ser Phe Arg Tyr 1 5 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> G4S DNA sequence <220> <221> CDS <222> (1)..(15) <400> 4 ggt ggc ggc ggt agc 15 Gly Gly Gly Gly Ser 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 Gly Gly Gly Gly Ser 1 5 < 210> 6 <211> 107 <212> DNA <213> Homo sapiens <400> 6 gcagctacag gtaaggggct tcctagtcct aaggctgagg aagggatcct ggtttagtta 60 aagaggattt tattcacccc tgtgtcctgt ccacaggtgt ccagtcc 107 <210> 7 <211 > 19 <212> PRT <213> Homo sapiens <400> 7 Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly 1 5 10 15 Val Gln Ser <210> 8 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> ANP-VH1-69 without leader <220> <221> CDS <222> (1)..(327) <400> 8 aac agc ttc cgg tac ggt ggc ggc ggt agc cag gtc cag ctg gtg cag 48 Asn Ser Phe Arg Tyr Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 1 5 10 15 tct ggg gct gag gtg aag aag cct ggg tcc tcg gtg aag gtc tcc tgc 96 Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys 20 25 30 aag gct tct gga ggc acc ttc agc agc tat gct atc agc tgg gtg cga 144 Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg 35 40 45 cag gcc cct gga caa ggg ctt gag tgg atg gga ggg atc atc cct atc 192 Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile 50 55 60 ttt ggt aca gca aac tac gca cag aag ttc cag ggc aga gtc acg att 240 Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile 65 70 75 80 acc acg gac gaa tcc acg agc aca gcc tac atg gag ctg agc agc ctg 288 Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 85 90 95 aga tct gag gac acg gcc gtg tat tac tgt gcg aga gac 327 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp 100 105 <210> 9 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Asn Ser Phe Arg Tyr Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 1 5 10 15 Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys 20 25 30 Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg 35 40 45 Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile 50 55 60 Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile 65 70 75 80 Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 85 90 95 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp 100 105 <210> 10 <211> 583 <212> DNA <213 > Artificial Sequence <220> <223> ANP-VH1-69 Cas9/BHR donor <400> 10 ctcgagcagc tacaggtaag gggcttccta gtcctaaggc tgaggaaggg atcctggttt 60 agttaaagag gatttattc acccctgtgt cctgtccaca ggtgtccagt ccaacagctt 120 ccggtacggt ggcggcggta gccaggtcca gctggtgcag tctggggctg aggtgaagaa 180 gcctgggtcc tcggtgaagg tctcctgcaa ggcttctgga ggcaccttca gcagctatgc 240 tatcagctgg gtgcgacagg cccctggaca agggcttgag tggatgggag ggatcatccc 300 tatctttggt acagcaaact acgcacagaa gttccagggc agagtcacga ttaccacgga 360 cgaatccacg agcacagcct acatggagct gagcagcctg agatctgagg acacggccgt 42 0 gtattactgt gcgagagaca cagtgtgaaa acccacatcc tgagagtgac aaaaaccctg 480 agggcgccgg cggaattcaa gcttcctagg cgccggcgag aaggcagctg tgccgggctg 540 aggagatgac aggggttatt aggtttaagg ctgtttaactc gag 583 <210> 11 <211> 19726 < 212> DNA <213> Mus musculus <400> 11 aagcttatct ctctgttgct cagactcatc taggaatttc agaaatttct gttctagcat 60 ctcttccagc ttttgtctcc aaccctcatt ctcttctttc tttttttttt taaattatat 120 gttctctgtc ttt ttaaaaa actttttaaa attaggtatt tatgtcattt acatttccaa 180 tgctatccca aaagtcccac ccacgctccc caacccacta tcccacccac ccactccccac 240 ttcttggccc tggcattcac agtgtactga gacatataaa gtttgcacaa ccaatgggcc 300 tctct ttcca ctgatggccg actaggccat cttctgatac atatgcagct agagacacga 360 gattctgggg gtactggtta gttcatattg ttgttccacc tatagggttg cagatccttt 420 tagctccttg ggtactttct ctagctcctc cattgggggc cctgtgatcc atccaatagc 480 tgactgtgag catccacttc tgtgtt tgct aggccccaga tagtctcaca agagacagct 540 atatctgggt cctttcagca aaatcttgct agtgtatgca acggtgtcag agtttggaag 600 ctgattatgg gatggatccc cggatatggc attctctagt tggttcatcc ttttgtctca 660 gctccaaact ttgtctctgt aact ccttcc atgggtgttt tgttcccagt tctaaggagg 720 ggcaaagtat ccacactttg gtcttcattc ttcttgagtt tcatgtgttt tgcaaattgt 780 atcttatatc ttgggtattc taagtttctg ggctaatatc cacttatcag tgagtacaca 840 ttgtgtgagt tcttttgtga ttgggttacc tcactcagta tgatgccctc caggtccatc 900 catttgccta ggaatttcat aa attcattc tttttaatag ctcagtagta ctccattgtg 960 tagatgtacc acattttctg tattcattcc tctgttgagg ggcatctggg ttctttccag 1020 cttctggcta ttataaataa ggctgctatg aacatagtgg agcatgtgac cttcttaccg 1080 gttgggacat cttct ggata tatgcccagg agaggtattg ctggatcttc cggtagtact 1140 atgtccaatt ttctgaggaa ctgacaaact gatttccaga gtggttagta ccagcttgca 1200 atcccaccaa caatgagagg agtgttcgtc tttctccaca tcctcaccag catgctgctg 1260 tcacctgaat ttttgatgct tagccattct gactggtgg aggtggaatc tcagggttgt 1320 tttgatttgt atttccctga tgattaagga tgctgaacat tttctcaggt gcttctcagc 1380 cattcagtat tctttaggtg agaattcttt gtttagctct aagccccatt tttttaatgg 1440 ggttatttga ttttctggag tccaccttct tgagtttttt tttccatttt ttattacata 1500 atttcctcaa ttacatttcc aatgctatcc caaaagtccc ccataccctc ccccccccaa 1560 ttccctaccc accccttccc atttttttgg ccctggcgtt cccctgtact ggggcatata ctag ctcctc cattgggagc 1800 cctgtgatcc atccaatagc tgactgtgag catccacttc tgtgtttgct aggccccggc 1860 atagtctcac aagagacagc tacatctggg tccttttgat aaaatcttgc tagtgtatgc 1920 aagggtgtca gcatttggaa gctgattatg gggtggatcc ctggatatgg cagtctctac 1980 atggtccatc cttttgtctc agctccaaac tttgtctctg taacttcttc catgagtgtt 2040 ttgttcccaa ttctaaggag gggcatagtg tccacacttc attcttcatt cttcttgagt 2100 ttcatgtgtt tagcaaattg tatcttatat cttgggtatc ctaggttttg ggctaatatc 2160 cacttatcag tgagtacata ttgtgtgagt tcctttgtaa atgtgtta cc tcactcagga 2220 tgacgccctc caggtccatc catttggcta ggaatttcat aaattcattc tttttaatag 2280 ctgagtagta ctccattgg taaatgtacc acattttctg tactcattcc tctgttgagg 2340 ggcatctggg ttctttatag gttctggcta ttataaata a ggttgctatg aacatagtgg 2400 agcatgtgtc cttcttaccg gttgagacat cttctggata tatgcccagg cgaggtattg 2460 ctggatcctc cggtagtact atgtccaatt ttctgaggaa ctgccagact gatttccaga 2520 gtggttgtac aagcctgcac tctcaccaac aatggaggag tgttcctctt tctccacatc 2580 cacgccagca tctgctgtca cctgaatttt tgatcttagc cattctgact ggtgtgaggt 26 40 ggaatctcag ggttgttttg atttgcattt ccctgatgat taaggatgtt gaacattttt 2700 ttcaggtgct tctctgccat tcggtattcc tcaggtgaga attctttgtt cagttctgag 2760 ccccattttt taatggggtt atttgatttt ctgaagtcc a ccttcttgag ttctttatat 2820 atgttggata ttagtcccct atctgattta cgataggtaa agatcctttc ccaatctgtt 2880 ggtggtcttt ttctcttatt gacggtgtct tttgccttgc agaaactttg gagtgagttc 2940 tttatatata ttggatatta gtcccctatc tgatttagga taggtaaaga tcctttccca 3000 atctgttggt gacctttttg tcttattgac ggtgtctttt gccttgcaga atctttgcaa 3060 ttttatgagg tcgcatttgt caattctcga tcttacagca caagtcattg ctgttctgtt 3120 caggaatttt tcctctgtgc ccatatcttc gaggctttta cctgctttct cctctatatg 3180 tttgagtgtc tctggtttaa tgtggagttc cttaatccac ttagatttga ccttagtaca 3240 aggagatagg aatggatcaa ttcgcattct tctacatgat aaccgctagt tgtgccagca 3300 ccatttgttg ataatgctgt cttttttcca ctggatggtt tttgctccct tgtctaagat 3360 caagtgacca taggtgtgtg ggttcatttc tgggtcttca attctatttc attggtctac 3420 ttgtctgttg ttataccagt accatgcaga ttttatcaca attgctctgt agtagagttt 3480 taggtcaggc atggt gatta caccagaggt tttttttatc cttgagcaga gtttttgcta 3540 tcctaggttt tgtgttattt cagatgaatt tgcagattgc cctttccagt tcgttgaaga 3600 attgagttgg aattttgatg gggattgcat tgaatctgta gattgctttg gcaatatagc 3 660 catttttaact atattgatcc tgccaatcca tgagcatggg agatctttcc atcttctcaa 3720 atcttcttta atttctttct tcagagactt gaagttcttg tcatacagat ctttcacttc 3780 cttagttaga gtcacgctaa ggtattttat attatttgg actattgaga agggtgttgt 3840 ttccctaatt tctttctcag cctgtttatc ctttgtgtac agaaaagcca ttgacttgtg 3900 ttagttaatc tcatatccag ctact tcact gaagcggttt atcaggttta ggagttctct 3960 ggtgtaattt ttagggtcac tcatatatac tatcatatca tctgcaaaaa gtgacatttt 4020 gacttcttcc tttccaattt gtatcccctt gatctccttt tgttgtcgaa ttgctctggc 4080 aag gacatca agtactatat tgaataggta gggagaaaat cggcaccctt gtctagtccc 4140 tgattttagt aggattgctt caagtttctc accatttact ttgatgttgg ctactggttt 4200 gctgttgaat gctttttatc atgtttaggt atgggccttg aattcctgat ctttccaaga 4260 cttttatcat gaaagggtgt tggattttgt caaatgcttt ctccagcctt tcattctgag 4320 gttgtgtctg tctttttccc tgaga tgggt ttcctgtaag cagcaaaatg ttgggtcctg 4380 tttgtgtagc ccgtctgtta ttctatgtct tttattggg gagttgagtc cattgatatt 4440 aagatatatt aaggaaaagt aattgttgct tcctattatt tttgttttta aagttggcat 4500 tct gttcttg tggctgtctt cttttaggtt tgttgaagga ttcctttctt gctttttcta 4560 ggtcgtggtt tccatccttg tattcatttt ttttctgtta ttatcctttg aaggactgga 4620 ttcatggata gataatgtgt gaatttggtt ttgtcttgga atacttttgt ttctccatct 4680 acggtaattg agagtttggc tgggtatagt agcctgggct ggcaattgtg ttgtcttagt 4740 gtctatataa tgtctgtcca ggatcttctg gctttcatag tctgt ggtga aaaatctggt 4800 gtaattctga taggcttgcc tttatatgtt acttgaattt ttcacttact gcttttaata 4860 ttctttcttt atttagtgca tttgttgttc tgattattat gtgtcggggag gaatttcttt 4920 tctggtccag tctatttgga gttctgtag g cttcttgtat gttcacgggc atctctttct 4980 ttaggtttgg gaagttttct tctataattt tgttgaagat atttgctggc ccttcaagtt 5040 gaaaatgttc attctcatct actcctatta ttcgtatggt tggtcttctc attgtgtcct 5100 ggatttcctg gatgttttga gttaggatct ttttgcattt tccattttct ttgattgttg 5160 tgcagatgtt ctctatgggaa tcttctgcac ctgatattct ctcttccatc tcttgtagtc 5220 tgttgctgat gctcgcatct atggttccag atttctttcc tagggtttct atctccagtg 5280 ttgccccact ttgggttttc tgtatagtgt ctacttccct tttagatct agtatggttt 5340 tgttcatttc catcacctgt ttgggtgtgt tttcctgttt ttctttaaag acttgcaact 5400 ctttagcaga gttctcctgt atttaagtga gttattaaag tccttcttga tgtccagtac 5460 cataattgtg agatatgcct ttaaatccaa gtctaggttt ttgggtgtgt tggggtgccc 5520 tggactggct gagttgggag tgctgcattc tgatgatggt gagtggtctt ggtttctgct 5580 agtaagattc ttacatctgc ctttcgccat ctggtaatct ctggagtca g ttgttaaagt 5640 tgtctctggt taaagcttgt tcctctcgtg attctgttat tctcttccag cagacctggg 5700 agactagctc tttcctgagt ttcagtggtc agagcactct ctgcaggcag gatttcctct 5760 ttcagggaag gtgcacagat atctggt gtt cagatttgcc tcctggcaga agatgatggc 5820 ctgaaacagg acctgtccca gaagctgtta gcttctgtag tcaacactgt cacctgtgca 5880 gactagtctc ggtggagtcc gggaaccaag atgtctcctg cagatgctct ggcattccct 5940 tctgggccgg gtgatcacct ctcctctggc agggaaggtg ccctggtgtc tggaacccga 6000 aaagggggct gcctcagaag ctctgtggct actgcctgtc ccagaagctg ttagcttctg 6060 tagtccacac tctcacctgt gcagactagt cttggtggag tctgggaacc aagatgtctc 6120 ccgcagatgc tccagccatt ctcctctttc tgttgcttat tttgacctat gaatcctgg 6180 acatatagtt ctagtgttgc ttgtaatctc ttttctaagc caaggaattt tttttatcta 6240 gggcacaatc ttttgagaag acatattaaa tcaagagaat aaatattgca agaccaataa 6300 atgataaggt atctattttc tttaaatcca tcgctgtcaa accattcaaa atatcctcac 6360 ataaagccaa aaagatattt attgtgtttc ccatcttagt tgagttcaag tcaatatttt 6420 ggtgccattt tgttgcagta aatctctaac acaaatatgc ctgggcaatg aaaacacaac 6480 tcagttaata t gaatacaga ttgttcagat ctaccactac actaccatct tcttcatcta 6540 agagacccct tagaacttgc agtttctcca ggccttgtgc ttctgcgctg cttttcttct 6600 tcttcctctt ctacattgct tctctcataa acctacttct ttttttccct ccttctgttc 6660 catcttccct tttatctgcc caatcattag ctctccttta ttttacaaat taaggtgtga 6720 agccggtttc taggaaatca cctgagtgct gacttgttcc a aaccaggag cttgttcttt 6960 gagaaaaatc aacaagatag ataaaccctt agccagacta accagagggc acagagacag 7020 tatccaaatt aataaagtca gaaatgaaag gaagacataa caatgaaata tatcttaaaa 7080 taattaatct gtttgtagac tattagcagt tgaaaatatt aaaatcatgt tctacaaacg 7140 tggaattatt attgataatt ttctcactgt gcttgaaatt agcattttct taatgtttaa 7200 cttcaaagag tttttgctat tttgaaatat taaacatata cttactgata aaataatttc 7260 cctcctaaca acactgataa tcttttttta agtaaactga ttattagaca atgtacacag 7320 atatataatg tgttttaaat actctcccac tgtcaggtgg tatcata tag ggcctttgaa 7380 tatattttta aatgtattat ttgtaatatt ttatggtctc tcctatgctt atttctgaaa 7440 gaatattttg tatgttttga aacaatttag tatttaacat tagatatagg atcctcagtt 7500 atggatagta ttaaatattc attaatgata tttttaaggt ataaa aggat atgaatataa 7560 aagtttaaca aattttatgt attatttgat tctaaaaata ctcaatatta ttaatatgtt 7620 tgatgtttaa aatgcattta aataataaaa acatttaaaa aaataaaatc aagaaatgag 7680 gttctaagca gaggtcaagg aaaatgagga atagaaaaat agtaaaaatc aatatgtcca 7740 tttatcaag gaaagctcct acatagacat tgcaccagat tagcaaata aga acagcat 7980 tatagacatt acccagaacc ttagtggttc tagaatgcca agataaaaca atctaacctt 8040 ctggatagta gggataaatg ttcctatatc atcagaattc actggtgccc tgaggatgtt 8100 accctgctaa ctgacaattc acaggacatc acatggattc tgataagttg cagaaaagag 8160 gagatgcatt caattggtcc tcctccttct aagctgcaat attaggtgca tccaatttgt 8220 gaacttcaat ttagattaca atagacatga ataatctgaa ttcatgtagt acatattttt 8280 gttttaatat gagttaccat tgttcagaaa attaaataca catgatcaca tattcctaca 8340 tagtgctgtt agtttttcac atctctggga caatattcca aatatctcct tcattagtga 8 400 aaatatcaac tactgtaaag cttagctaac atgcctttgc aggaataaga acatcctgga 8460 ttgaaagcta cacagggaga tgtaaaactt tctaagcaca cacattctcc atccattagg 8520 atcatggtcc atgagatttt tctctctctc ttcttcccat taaatgcatg tacatgcagg 8580 ttgggaaaca gattgtgttg cagaatacat ttgcttgatt tccacttcct tctcaatgca 8640 aat attttg aagtgttaat tttgctgtga gtaccacagt ggttcttgct ctttctgttg 8700 actcctgtct gtgaatgttc caggaattca cacatggaca cacgtggggc tgcatctgag 8760 ctccagactc actgttgtcc ttctgtcctc agctgctctg gcccaggcac agcctcgt ga 8820 attcaacaaa gaccctgatc tctcttgttt acacctcatt acaaatggga actgttagag 8880 gtggacccaa ctgcatttcc atgaggaaag cacatgagtt tgagagggtc gttgatgata 8940 aggtagaaac aactttaatt cataggctga gatatcagtc atcacctcca gataaacaag 9000 agccatttct tcctgcatct gagccctgta agcacactag ctttaggaat atgttactgc 9060 tgaagtcaga ttgggcaact tcatagtata caatagaaaa tctacctgca gatgagttca 9120 gaaccagcag ggggcacaat ggggccaaga atccctagca gagagatgg gtgtgtgg c 9180 aggggactct gcatcctctg tggtttcctt tcttaactta catgtacctg tagtgattga 9240 catgtaacgt ttccacgctc aaacactgtg aagatacttt gctaaacact tcaaagattt 9300 atgtttctt gatgtgtgca tgtgtgtatt cttttttgtt tttagac aca gggtttctct 9360 gtgtagtcct ggctgccctg gaactcactc tgtagaccag gctggcctcg aactcagaaa 9420 tctgcctgct tctgcctccc aagtgctgaa gttaaagaca tgtgccacca ttgcctggcc 9480 atgtgtgtat tcttgatgca ctcttctgtt gacagataca cagtttattt ccataattta 9540 tttatgtga tggtgctgca ataatcactt atgtacaaat gtttctgaag tatatttagt 96 00 tttggtcatt tgggtgatta ttttttctt tctagtatat agcattttgg aaaggtagat 9660 attaattgta tgtatgggaa ggaggctgta aattctaata acttagctgc ttttgaaatt 9720 tgtcctcaat tctatcatcc ttgtaaccac cttaaatcca tctatt agcc ttgtcacaag 9780 tgagccactg tctcaggctg caaatctttt tatagattag gtcgtgatgt tacatccaca 9840 gcctctgcac aatgctcagg ggtgggaat gggatgaatt ccctcagaca gcattaggac 9900 ttggatctca gcagactgat tcttgaccca aatgtctctt cttctctagc aggagtaagt 9960 ccttatctaa gatgtactct gctcatgaat atgcaaatca attgagtcta tggtggtaaa 10020 tatagggatg t ctacacccc tcaaaaactt aagatcactg tcgtcttcac agtcacagga 10080 gtacacagga catcaccatg tgttggagct gtatcatcct cttcctgtta gcaacagctg 10140 cacgtaaggg gcttacagta gcaggcttga ggtctggcca tacactcatg tgacaatgac 1020 0 atccactctg tccttccctt cacaggtgtg cactcccagg tccagctgca gcagtctggg 10260 gctgagctgg tgaggcctgg ggcctcagtg aagatttcct gcaaggcttt tggctacacc 10320 ttcacaaacc atcatataaa ctgggtgaag cagaggcctg gacagggcct ggactggatt 10380 ggatatatta atccttataa tgattatact agctacagaa ccagaagttc aagggcaagg 10440 ccacattgac tgtagacaaa tcctccagca cagcctatat ggagcttagc agcctgacat 10500 ctgaggactc tgcagtctat tactgtgcaa gacacagtgc tacaaacaca tcctgagtgt 10560 gtcagaaacc ctggaggaga agcaagcaga gctggaatgg agatgacaga aagattatca 10620 tttagacttg ctcagaaaga gaaattttga atgcccattt attgcctctt ccttacagta 10680 ctatagtgcc tgtttttgtt gacattttca aactaatttc caaagtcact accacaattt 10740 acaatcacat aaaaagcaag caaggataac attattttct gtgcttactt gccatttata 10800 ttcttgctta ttctcatctc actgaggtca tattgggaca ttaaatttct ggggttactt 10860 tttattaaaa atttttcatt attcattc ac tttacatcct tctagtcttc ctctcacaca 10920 tgccctatcc ctttctcctc tgagaggatg gagccctccc taccctcgta tccccttacc 10980 caggcacatc aagtgtctgc agtactagga atattctctg tcaatgctgc cagacaaggc 11040 agacaagtta gg ggatcagg attcacagga aggcaacagc ttgagggaca gcccccactg 11100 aagttatgg tggattcaca tgaagactga gttgcacatc tgctacatat attcaggggt 11160 cctatttaca gctcaagtag actcttgttg gtggtttagt ctcttagaac cccaagtgtc 11220 caggttagtt gactctgtgg gtcttccttt ggagttccta tcccctccag atccctcagt 11280 tcttctccca actcttccat aagacacccg taggtccatc caat gtttgg ttttgggttt 11340 ttctgcatct gcttcagtca gctgctgggt ggagcatctc tgaggataat tatgagaagc 11400 tcttatgtgc aagcataaca ggatatcatt attagtgtca gggactggtg cttggccatg 11460 ggatgggtct caagtttggt cagttat tg gccattccca cagtctctga taatctttgt 11520 ccctgcattt cttgtagaca ggaaaaatat tgggttgaaa gttttgtggg tgggttggcg at catacaga actgcatgtg 11760 tgcaaccaca cagaacaagg ctatctatca gaggcctacc ataccaggac catcaaggtt 11820 caccttactc ccaatactga ctacaaaaag aacatcaagg accaatgcag tctatatgga 11880 taaacacact tgaaagaaca caaacaagat tgagggcaac atga cacctc caaagcatac 11940 ctaaccgagt acagcatgcc ctggatatcc taacacaatc aaaacacaag aaagttacct 12000 taaatccagt cttataaagg tgatgaaggc ctttaaatag gaaatgaatt aatccttagg 12060 ataatacagg acaatacatt cgaacagata gaggtcttta ggaggaaaga aataaatccc 12120 tcaaagacat acatgaaaat acaattaaac aggtgaaagt aataactaca atggtgtaag 1218 0 acctaaaaat ggaaatagaa gcaataaagt aacacaaact tagaatcttg aaggtggaaa 12240 acctagagaa caggaatact agatgcaagg atgatatctt ctaggtccat ccatttgctt 12300 gcacaattta tcatgtcctt gcttttaata gttgaacagt atttcattgt ttaaatgaac 1236 0 cacatgttct gtctccattc tctggatgag ggggtgagca agtttttcca cattctggct 12420 attacaaata gagctgctat gaacctagta gaaaacatat cctgtgtatg gtggagagtt 12480 ttggagtata tcaccaagag tgttatagct gggtcttcat gtagaactat tcctaatttt 12540 ctgagaaatc ccaagtcaga tttctagaat ggttgttcaa gtgttcactc caaccatcaa 12600 tggaggactg ttttccttgc cagcatgtgc tgtattttga gttttgatc ctagccagtt 12660 ttatcctgca tttcacactt agatatggac tatggtacag gacagagaga aaccaacctt 12720 ctactcacca ggatattcta cctgctacca atttattta t ttattatattt atttatttat 12780 ttattatattt atttatttat attagagaac aacaccatgc agtttagaag aagtactaag 12840 acgtcagtga tgttatactg tgcctaacct tgcattgtac aatctcagct ttcaggtaag 12900 acagtgcatg actcttatgc agtgccaact gttttctgat tgtatttatg gtctattgcc 12960 taggaatgac ctcctctcaa ataaacatgg tcaaaagccc atggcctgag atgacagagc 13020 ccctagtaga ccctagttgt atttctgaag tttagatatc ataatgactt ataaatactt 13080 atgtttatac aatagattag agctgctctc agccatgacc aaggagcttc tgtgttcaat 13140 gaataatgat tgatgcagac attcgtgagt ggtcaaagtg gtgagaatga ttagaga gtc 13200 ctcagccaca caagcgttaa tgatatgaac tttccaatat attaactgta ttaatgaata 13260 aatgcagaca tcatatgaga tctcattagt agttcttagg tattgcattt ttatatacaa 13320 ttatgcatat cagtacatta tagtgtataa aggaaattgt ctagcataat agagaaaaat 13380 aggacagtca agaaacaaaa gagtagaaat tatgggtgaa atatgcagtg tgaaatattt 13440 acatgaaaat tttaaccata tgta aaattg ttatttttgt ttttcagaat gagtttgctc 13500 attctttgac attttattc ctgtgtgaaa tatatcagga tcatatgtat cccattctga 13560 tggtctgact tccactggga atttccaata tatctcttcc aactaactga ccagtttctt 13620 t ttttcttat tttctctctt tctcgttttg ttttgctttg ttttgttttt caagacaggg 13680 tttctctgtg tagctctggc tgtcctggaa ctcactttgt agatcaggct ggcttcgagc 13740 tcataaatcc acttgcctct gcctcctgag tgctgggatt aaaggagtgg ctaccacgcc 13800 cggctagttt ttttttttct tataagaaca acatttactg gatggtcact tacatattca 13860 gaggttcagt caattattat caagg cagaa gcatggcagt ggtccagtag tcatggcact 13920 ggggaaggag ctgagagatc tacatcttgc tccaaaggga aagaggaata gtctgacttc 13980 catgtgtttc agaggagggt ttcatttccc acccccacag tgacacactt cctccaacac 14040 ggccccacat cc taatattg ccactcttgg atcaagcata ttcacaccac aaaggaaagt 14100 ttagagataa acattaagaa aattaatgaa gtcattttat cttatatgct caacatgact 14160 agtacttaaa accataattt tacatgtaca atatttcatg gcataacata ttttttatat 14220 ttttattaga tattttcttt atttatattt caaatgtgat accctttccc aattcccctc 14280 caaaaatccc ctatgccttc ccctcatagc cagctcccaa acc cacccac tcctgctttc 14340 tggtcctggc attcccctat actggggcat aaaaccttca caggaccaag tgcctcttct 14400 ccattgatgg ccaattaggc catcctctgc tacatatgca gctagagcca tgagttccac 14460 catgtgtttt ctttgattgg tggt ttagtt ccagggagct ctgggggtat tggttagttc 14520 atattgttcc tcctatgggg ctgcaaaccc tttcagcccc ttgggtattt tttctagctc 14580 cttcattggg gaccctgtgc tccatccaat ggatgagtga gcctccactt ttgtatttgt 14640 caggaactgg cagagtctct caggagacaa ttatatcagg ctcctgtcag caaaatctcg 14700 ttggcatctg caatagtgtc tgggtttggt ggttgttta t gggatggatt tctgggtggg 14760 gcagtctctg gattgtcatt cctttagtct ctgcttccac ctttgtcttt gtaactccat 14820 ccatgggtat tttgttcccc cttcaaagaa ggatcaaaat atccacactt tagtcttcct 14880 tcttcttgag tctcatgtgt ttt tcaaatt gtatcttggg tattctgagc ttctaggcta 14940 atatccactt atcagtgagt gattatcatg tctgttcttt tgtgattgag ttacctcact 15000 tagcatgata tcctccaggt ctatccattt gtctaagaat ttcataaagt cattgtcttt 15060 aatagctgca tcgtactcaa ttgtgtaaat gcaccacatt ttctttatcc attcctctgt 15120 tgaggacac ttggtttttc ccagcttctg gttattataa ataaggctg c tatgaacata 15180 gtggaacatg tgtccttagt acatgttgga acatcttctg ggtatatgcc caggagtggt 15240 attgctggat cttctggtgg tactatgtcc aaatttttgg ggaaccatca aactgatttc 15300 ctgagtggtt gtacaagctt gcaatcccac accagcaata gtggaatgtt catctttgtc 15360 caagtccttg ccagcatctg ctgtcacctg agtttttgat cttagccatt cttactggtg 15420 tgaggtggaa tcttggggtt gttttgattt gcatttccct gatgtttaag ggttttgaac 15480 atttttaggt gcttattaga catttggtat tcctcagttt agaaatcttt gtttagctct 15540 gtaccacatt tttgaatagg gttatttggt tttctggagt ctaacttctt gagtt ctttg 15600 tacatattgg atattagccc tctatcagat ttagaattag taaggatctt tccccaaact 15660 gttggtggtt cttttgtctt attgacagtg tactttgcct tagagaagct ttgcaatttt 15720 atgaggtccc atttgtcaat tcttgatctt atagtacaag ccattggtct tttgttcagg 15780 aatttttccc atgtgtccat atgttcaagg catttcccca ctttctccac tacaagtttt 15840 agtgtctctg gttttatgtg gaggtccttg atccacttag atttgagctt tgtacaagga 15900 gataagaatg gatagattca cattcttcta catgctctct gccagttgag ctagcaccat 15960 ttgttgaaaa tgctgtcttt ttttccccc actggatggt ttttagctct tttggccaag 16020 atcaagtgac cattggtgtg tgggttcatt tcttggtctt caattctagt tcactgactt 16080 acctgtttgt cactgtacaa ggaccatgca gcttttttca caattgctct gtagtacagc 16140 ttgaggtctg ggatggtgat tctaccagag agattcttttgt acttgtga ataatttttg 16200 ctatcatagg atattttttt atttcagatg aatttacaaa ttgctctttc taactctgtg 16260 aacaattgag ttggaatttt gattgtgatt gctttgaata ctcaagatat aatttacaaa 16320 acacatgaaa cttaacaagg actactaaag tgcagatact tcgatccttc ttagaagggg 16380 gaacaaaata cccatagatg gagttacaga gacaaagttc ggagcagaga ctataggaac 16440 gaccatccag aggtccacct gg ggatccat catgtaaaca accacccaaa acagacacta 16500 ttgtggatgc caagaagaac ttgctgacag gagtctgata tagctgtctc ttgagaggct 16560 ctgccagggc ctcagaaagt ggaggctcac agccatccat tggatggagc acagggtccc 16620 caatgaagga g ctagagaaa gtactcaagg agctgaaggg gtttgcagcc ccataggagg 16680 aacaacaata tgaactaacc agtaccccca gagctccctg ggactaaacc accaatcaaa 16740 gaaaacacat ggagggactt gaagctcttg ctgcatttat agcagaggat ggcctagatg 16800 gtcatcaatg ggaggagagg tcaatggtcc tgggaaggtt ccatgcccca gtatagggga 16860 atgccagggc caggaagcag gagtgggtgg gctggggatc agggagggggg agatgatagg 16920 gcattttcag tggggaaact agggaaagagg ataacattta aaatataaat aaagaaaata 16980 tctaattaaa aaggattacc tatgtgcatg ggagctcatg agcagcaggg gtcactctaa 17040 ggccaataat ccacatagag cgatgagctg tgtgtgaaca ggactctgta tcctctgtgg 17100 tttcctttct taagtgtatt aactgatctg tccagctggtg attgacatgt gatgtctcca 17160 tgctcaagcc cagtaaagat tctctgttaa ataccttaca gacttatgtt tacttgtttt 17220 tatttgcttt tcatattttt ttaaaaagtc atacaatgta ttctaataac tcattctccc 17280 atctccaatt tattctaagt tttt cttaac tcatccaacc acacactttt taattctgat 17340 aaagcacccc cccccccaaa aaaaaaccca accaaccaaa aaaaaaaaaa gccaaggaat 17400 ttaaaagggg attgaaagca aataaaaact aaacaaaaaa gtaaaaacta cacacaca 17460 cac acacaca cacacacaca cacacacaca cacacactca cacacacaca cacacaccac 17520 acacacacac acccatgcac gaacacacac acacacacac acacacacac acacacacac 17580 acacacacac acacatggaa tccagtaaaa ccacaactct ttacccatga tacacaggaa 17640 aatataagtc aaacaaacag aatggaagaa ggtggtatta taaaaatgtc tgcacaaata 17700 ccattaagtt cattttcttg ttggctacca actgctaagc ctgtctcc ct tgattaattg 17760 tgcttatcat cccctatgaa ctccattgga ggacactaat ttttccttct gtctccagga 17820 attgaagtgt tgcagaactc tcagtagctt tatttacctg cacaatacag cctctaatcc 17880 aaccagtgaa aattacaca tgagagactt ccaa atgaaa gaacaggtaa agttgtctac 17940 tggcaagctt agtaatatca tgtaaatgcc ttagaattta atgacatatg tcatcctctg 18000 aggttaataa atccattttg gtgcatatat accctgaact caccactaac ataatacaac 18060 aattaaaaaa ttccaacatg gatgcagagg aatccctgag ggacatttgt tgatttgtga 18120 gcacaatata attatttttt gggggggaaa tgtctgaatg ttaactcttt accagtgata Atta tagcat 18360 atttcatatg caattgaaaa tttcaaagaa tgaaaatcct tatgaaatat agaaataaca 18420 actttactta tgtacatata cttcatagta caatttttac actgtgcata tttctcctgt 18480 aacatctggt tcctcctatt ttcctttatt ctcctagaca atttcactga tacaatctca 18540 tgtttttgta taaatagttg tatataacta ttaaatacat aagctgttaa tgagtcttca 1860 0 ttaatgtctg tgattttttt attgtcttaa ttaatactat tatctctaat tgcatccaca 18660 ttttcaaaag caatgtaaat ttcttactca tttctgttca aaaacttctg ttgttgtatc 18720 attaccatgc cttagtgata aaatcctttc ttgacacatc tatagctatt gctataattt 18780 agttattgat gatcctcctg caataatcat tgataggtaa atattttaag cacttttaact 18840 tttagtcatt ttagtgagat ttgaagtagt atataacctg ttggaaaggc aaatattaat 18900 tccatatatg tgaaagaaga cgctaaaact aaaaacatta gccactttta gatatcttct 18960 ccttcttctt cttcttcttc ttcttcttct tcttcttctt cttcttcttc ttcttcttct 19020 tcttcttctt cttcttttct tcttcttctt ctccttctcc ttctccttct ccttctcctt 19080 ctcctcttcc tcctccttcc ttccttcctt ccttccttcc ttccttcctt ccttccttcc 19140 ttccttcctt cctt ccttcc ttccttcctt ccttccttcc ttccttcctt ccttccttcc 19200 ttcctttctt tctttctttc tttctttctt tctttctttc tttctttctt tctttctttc 19260 tttctttctt tctttctttc tt tctttctt ctcctcctcc ttctttttcc ttctccttcc 19320 ccttcacctt ccccttcctt cctctttccc ttccccttct ccttctcctc aatctacaat 19380 ctgttaacat attaacatgt cccagagtag agcaacagac tcaggtcaaa catctactga 19440 gaaattt gcc catgtagtta acatctacag catctgtcta ggggttacaa aaagtctatg 19500 ggatacaatt cctcagaaag gaataggatt tggacctgag catactgctg cctaacacat 19560 gaaatggcag ttcttctcca gctggactag gtccttaact aagaaatgca ctgctcatga 19620 atatgcaaat tacccaagtc tatggcagta aatacagaga tgtccacacc ctgaagacaa 19680 cctatgaaca atgttctctc cacagtccct gaagacactg attcta 19726 <210> 12 <211> 7928 <212> DNA <213> Mus musculus <400> 12 ctgagcattg cagactaatc ttggatattt gtccctgagg gagccggctg agagaagttg 60 ggaaataaac tgtctaggga tctcagagcc tttaggacag atta tctcca catctttgaa 120 aaactaagaa tctgtgtgat ggtgttggtg gagtccctgg atgatgggat agggactttg 180 gaggctcatt tgagggagat gctaaaacaa tcctatggct ggagggatag ttggggctgt 240 agttggagat tttcagtttt tagaataaaa gtattagctg cggaatatac ttcaggacca 300 cctctgtgac agcatttata cagtatccga tgcataggga caaagagtgg agtggggcac 360 tttctttaga tttgtgagga atgttccaca ctagattgtt taaaacttca ttt gttggaa 420 ggagagctgt cttagtgatt gagtcaaggg agaaaggcat ctagcctcgg tctcaaaagg 480 gtagttgctg tctagagagg tctggtggag cctgcaaaag tccagctttc aaaggaacac 540 agaagtatgt gtatggaata ttagaagatg ttgcttttac t cttaagttg gttcctagga 600 aaaatagtta aatactgtga ctttaaaatg tgagagggtt ttcaagtact cattttttta 660 aatgtccaaa attcttgtca atcagtttga ggtcttgttt gtgtagaact gatattactt 720 aaagtttaac cgaggaatgg gagtgaggct ctctcataac ctattcagaa ctgactttta 780 acaataataa attaagtttc aaatattttt aaatgaattg agcaatgttg agttggagtc 84 0 aagatggccg atcagaacca gaacacctgc agcagctggc aggaagcagg tcatgtggca 900 aggctatttg gggaagggaa aataaaacca ctaggtaaac ttgtagctgt ggtttgaaga 960 agtggttttg aaacactctg tccagcccca ccaaaccgaa agtccaggct gagcaa aaca 1020 ccacctgggt aatttgcatt tctaaaataa gttgaggatt cagccgaaac tggagaggtc 1080 ctcttttaac ttattgagtt caacctttta attttagctt gagtagttct agtttcccca 1140 aacttaagtt tatcgacttc taaaatgtat ttagaattca ttttcaaaat taggttatgt 1200 aagaaattga aggactttag tgtctttaat ttctaatata tttagaaaac ttcttaaaat 126 0 tactctatta ttcttccctc tgattattgg tctccattca attcttttcc aatacccgaa 1320 gcatttacag tgactttgtt catgatcttt tttagttgtt tgttttgcct tactattaag 1380 actttgacat tctggtcaaa acggcttcac aaatcttttt caagaccact ttctgagtat 1440 tcattttagg agaaagactt tttttttaaa tgaatgcaat tatctagact tatttcagtt 1500 gaacatgctg gttggtggtt gagaggacac tcagtcagtc agtgacgtga agggcttcta 1560 agccagtcca catgctctgt gtgaactccc tctggccctg cttattgttg aatgggccaa 1620 aggtctgaga ccaggctgct gctgggtagg cctggacttt gggtctccca cccagacctg 1680 ggaatgtatg gttgtggctt ctgccaccca tccacctggc tgctcatgga ccagccagcc 1740 tcggtggctt tgaaggaaca attccacaca aagactctgg acctctccga aaccaggcac 1800 cgcaaatggt aagccagagg cagccacagc tgtggctgct gctcttaaag cttgtaaact 1860 gt ttctgctt aagagggact gagtcttcag tcattgcttt agggggagaa agagacattt 1920 gtgtgtcttt tgagtaccgt tgtctgggtc actcacattt aactttcctt gaaaaactag 1980 taaaagaaaa atgttgcctg ttaaccaata atcatagagc tcatggtact ttgaggaaat 2040 cttagaaagc gtgtatacaa ttgtctggaa ttatttcagt taagtgtatt agttgaggta 2100 ctgatgctgt ctctactt ca gttatacatg tgggtttgaa ttttgaatct attctggctc 2160 ttcttaagca gaaaatttag ataaaatgga tacctcagtg gtttttaatg gtgggtttaa 2220 tatagaagga atttaaattg gaagctaatt tagaatcagt aaggagggac ccaggctaag 2280 aaggcaatcc t gggattctg gaagaaaaga tgtttttagt ttttatagaa aacactacta 2340 cattcttgat ctacaactca atgtggttta atgaatttga agttgccagt aaatgtactt 2400 cctggttgtt aaagaatggt atcaaaggac agtgcttaga tccgaggtga gtgtgagagg 2460 acaggggctg gggtatggat acgcagaagg aaggccacag ctgtacagaa ttgagaaaga 2520 atagagacct gcagttgagg ccagcaggtc ggctggacta actctccagc cacagtaatg 2580 acccagacag agaaagccag actcataaag cttgctgagc aaaattaagg gaacaaggtt 2640 gagagcccta gtaagcgagg ctctaaaaag cacagctgag ctgagatggg tgggcttctc 2700 tgagtgcttc taaaatgcg c taaactgagg tgattactct gaggtaagca aagctgggct 2760 tgagccaaaa tgaagtagac tgtaatgaac tggaatgagc tgggccgcta agctaaacta 2820 ggctggctta accgagatga gccaaactgg aatgaacttc attaatctag gttgaataga 2880 gctaaactct actgcctaca ctggactgtt ctgagctgag atgagctggg gtgagctcag 2940 ctatgctacg ctgtgttggg gtgagctgat ctgaaatgag atactctgga gtagctgaga 30 00 tggggtgaga tggggtgagc tgagctgggc tgagctagac tgagctgagc tagggtgagc 3060 tgagctgggt gagctgagct aagctggggt gagctgagct gagcttggct gagctagggt 3120 gagctgggct gagctggggt gagctgagct gagctggggt aagctggggt gagctggggt 3180 gagctgagct gagctggagt gagctgagct gggctgagct ggggtgagct gggctgagct 3240 gggctgagct gggctgagct ggggtgagct gagctggggt gagctgagct gagctggggt 3300 gagctgagct gagctggggt gagctggggt gagctgagct ggggtgagct gagctgagct 3360 ggggtgagct gagctggggt gagctgagct gagctggggt gagctgagct gagctgagct 3420 gag ctgagct ggggtgagct gagctgagct gagctggggt gagctggggt gagctgagct 3480 gagctggagt gagctgagct gggctgagct ggggtgagct gggctgagct ggggtgagct 3540 gagctgagct gagctgagct ggggtgagct gagctgagct ggggtgagct gagctggggt 3 600 gagctgggct gagctgagct gagctgagct gagctgagct gagctgagct gagctgagct 3660 gagctgagct gagctgagct gagctgagct gagctggggt gagctgagct gagctgggct 3720 gagctggggt gagctgggct gagctgggct gagctgggct gagctggggt gagctgagct 3780 ggggtgagct gagctgagct gggctgagct gagctgagct ggggtgagct gagctgagct 3840 ggggtgagct gag ctgagct gagctggggt gagctgagct gagctgggct gagcagggct 3900 gagctggggt gagctgagct gagctggggt gagctgggct gagctgggct gagctgagct 3960 gagctgggct gagctgggct gagctgggct gagctgggct gagctgggct gagctggggt 4020 gagctgagct gg ggtgagct ggggtgagct gagctggggt gagctgagct ggggtgagct 4080 gagctgagct ggggtgagct gagctggggt gagctgagct gagctggggt gagctgagct 4140 gagctggggt gagctgagct agggtgaact gggctgggtg agctggagtg agctgagctg 4200 aggtgaactg gggtgagccg ggatgttttg agttgagctg gggtaagatg agctgaactg 4260 gggtaaactg ggatgagct g tggtgagcgg agctggattg aactgagctg tgtgagctga 4320 gctggggtca gctgagcaag agtgagtaga gctggctggc cagaaccaga atcaattagg 4380 ctaagtgagc cagattgtgc tgggatcagc tgtactcaga tgagctggga tgaggtaggc 4440 tgggatga gc tgggctagct gacatggatt atgtgaggct gagctagcat gggctggcct 4500 agctgatgag ctaagcttga atgagcgggg ctgagctgga ctcagatgg ctagactgag 4560 ctgtactgga tgatctggtg tagggtgatc tggactcaac tgggctggct gatgggatgc 4620 gccaggttga actaggctca gataagttag gctgagtagg gcctggttga gatggttcgg 4680 gatgagctgg gaaaagatggcggacca tgaactgggc tgagctgggt tgggagacca 4740 tgaattgagc tgaactgagt gcagctggga taaactgggt tgagctaaga atagactacc 4800 tgaattgtgc caaactcggc tgggatcaat tggaaattat caggatttag atgagccgga 4860 ctaaactatg ctgagctgga ctggttggat gtgttgaact ggcctgctgc tgggctggca 4920 tagctgagtt gaacttaaat gaggaaggct gagcaaggct agcctgcttg catagagctg 4980 aactttagcc tagcctgagc tggaccagcc tgagctgagt aggtctaaac tgagttaaaa 5040 atcaacaggg ataatttaac agctaattta acaagcctga ggtctgagat tgaatgagca 5100 gagctgggat gaactgaatg agtttcacca ggcctggacc agttaggcta ggacctcgtt 5160 ctatagaggc agactgtgtg ctacagtgga gtttcaagat gattccatga gtcctccccg 5220 cccccaacat aacccacctt cctcctaccc tacacgcctg tctggtgtgt aaatcccagc 5280 tttgtgtgct gatacagaag cctgagcccc tcccccacct ccacctacct attactttgg 5340 gatgagaata gttctcccag ccagtgtctc agagggaagc caagcaggac aggcccaagg 5400 ctacttgaga agccaggatc taggcctctc cctgagaacg ggtgttcatg cccctagagt 5460 tggctgaagg gccagatcca cctactctag aggcatctct ccctgtctgt gaaggcttcc 5520 aaagtcacgt tcctgtggct agaaggcagc tccatagccc tgctg cagtt tcgtcctgta 5580 taccaggttc acctactacc atatctagcc ctgcctgcct taagagtagc aacaaggaaa 5640 tagcagggtg tagagggatc tcctgtctga caggaggcaa gaagacagat tcttacccct 5700 ccatttctct tttatccctc tctggtcctc agagagtca g tccttcccaa atgtcttccc 5760 cctcgtctcc tgcgagagcc ccctgtctga taagaatctg gtggccatgg gctgcctggc 5820 ccgggacttc ctgcccagca ccatttcctt cacctggaac taccagaaca acactgaagt 5880 catccagggt atcagaacct tcccaacact gaggacaggg ggcaagtacc tagccacctc 5940 gcaggtgttg ctgtctccca agagcatcct tgaaggttca gatgaatacc tggtatgcaa 600 0 aatccactac ggaggcaaaa acaaagatct gcatgtgccc attccaggta agaaccaaac 6060 cctcccagca ggggtgccca ggcccaggca tggcccagag ggagcagcgg ggtggggctt 6120 aggccaagct gagctcacac cttgaccttt cattccagct gtcgcagaga tgaac cccaa 6180 tgtaaatgtg ttcgtcccac cacgggatgg cttctctggc cctgcaccac gcaagtctaa 6240 actcatctgc gaggccacga acttcactcc aaaaccgatc acagtatcct ggctaaagga 6300 tgggaagctc gtggaatctg gcttcaccac agatccggtg accatcgaga acaaaggatc 6360 cacaccccaa acctacaagg tcataagcac acttaccatc tctgaaatcg actggctgaa 6420 c ctgaatgtg tacacctgcc gtgtggatca caggggtctc accttcttga agaacgtgtc 6480 ctccacatgt gctgccagtg agtggcctgg gctaagccca atgcctagcc ctcccagatt 6540 agggaagtcc tcctacaatt atggccaatg ccacccagac atggtcattt gctccttgaa 660 0 ctttggctcc ccagagtggc caaggacaag aatgagcaat aggcagtaga ggggtgagaa 6660 tcagctggaa ggaccagcat cttcccttaa gtaggtttgg gggatggaga ctaagctttt 6720 ttccaacttc acaactagat atgtcataac ctgacacagt gttctcttga ctgcaggtcc 6780 ctccacagac atcctaacct tcaccatccc cccctccttt gccgacatct tcctcagcaa 6840 gtccgctaac ctgacctgtc tggtctcaaa cctggcaacc tatgaaaccc tgaatatctc 6900 ctgggcttct caaagtggtg aaccactgga aaccaaaatt aaaatcatgg aaagccctcc 6960 caatggcacc ttcagtgcta agggtgtggc tagtgtttgt gtggaagact ggaataacag 70 20 gaaggaattt gtgcgtactg tgactcacag ggatctgcct tcaccacaga agaaattcat 7080 ctcaaaaccc aatggtaggt atcccccctt cccttcccct ccaattgcag gacccttcct 7140 gtacctcata gggagggcag gtcctcttcc accctatcct cactactgtc ttcatttaca 7200 gaggtgcaca aacatccacc tgctgtgtac ctgctgccac cagctcgtga gcaactgaac 7260 ctgagggagt cagccacagt cacctg cctg gtgaagggct tctctcctgc agacatcagt 7320 gtgcagtggc ttcagagagg gcaactcttg ccccaagaga agtatgtgac cagtgccccg 7380 atgccagagc ctggggcccc aggcttctac tttaccca gcatcctgac tgtgacagag 7440 gaggaatggacc actggaga g acctatacc tgtgttgtag gccacgaggc cctgccacac 7500 ctggtgaccg agaggaccgt ggacaagtcc actggtaaac ccacactgta caatgtctcc 7560 ctgatcatgt ctgacacagg cggcacctgc tattgaccat gctagcgctc aaccaggcag 7620 gccctgggtg tccagttgct ctgtgtatgc aaactaacca tgtcagagtg agatgttgca 7680 ttttataaaa attagaaata aaa aaaatcc attcaaacgt cactggtttt gattatacaa 7740 tgctcatgcc tgctgagaca gttgtgtttt gcttgctctg cacacaccct gcatacttgc 7800 ctccaccctg gcccttcctc taccttgcca gtttcctcct tgtgtgtgaa ctcagtcagg 7860 cttaca acag acagagtatg aacatgcgat tcctccagct acttctagat atatggctga 7920 aagcttgc 7928 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> GLSG <400> 13 Gly Leu Ser Gly 1 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker - GGGGS <400> 14 Gly Leu Ser Gly Ser Gly 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> linker - GLSGLSGS <400> 15 Gly Leu Ser Gly Leu Ser Gly Ser 1 5 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> GLSGLSGLSG linker <400> 16 Gly Leu Ser Gly Leu Ser Gly Leu Ser Gly 1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> GLSGGSGLSG <400> 17 Gly Leu Ser Gly Gly Ser Gly Leu Ser Gly 1 5 10 <210> 18 <211> 20 < 212> DNA <213> Artificial Sequence <220> <223> 5' detect VH1-69 FR3 <400> 18 acagaagttc cagggcagag 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223 > 3' up detect spec <400> 19 tgtccactgg gttcgtgcct t 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5' down detect spec <400> 20 cagtatcagc ccgtcatact t 21 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 3' VH1-69 overlap detect <400> 21 taacccctgt catctcctc 19 <210> 22 <211> 23 <212> DNA < 213> Artificial Sequence <220> <223> 5' DNA Target (PAM) <400> 22 ggatcctggt ttagttaaag agg 23 <210> 23 <211> 42 <212> RNA <213> Artificial Sequence <220> <223> 5' VH1-69 Cas9 crRNA <400> 23 ggauccuggu uuaguuaaag guuuuagagc uaugcuguuu ug 42 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 3' DNA Target (PAM) <400> 24 gacaaaaacc ctgagggaga agg 23 <210> 25 <211 > 42 <212> RNA <213> Artificial Sequence <220> <223> 3 VH1-69 Cas9 crRNA <400> 25 gacaaaaacc cugagggaga guuuuagagc uaugcuguuu ug 42 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 5' up detect VH1-69 <400> 26 ctgtgaaata ccctgcctc 19 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3' up detect spec <400 > 27 tgtccactgg gttcgtgcct t 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5' down detect spec <400> 28 cagtatcagc ccgtcatact t 21 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3'down detect M1116(h70) <400> 29 ccccctcttg ctctctttct 20 <210> 30 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> VH1-69 MreI del Joiner Oligo <400> 30 gtgaaaaccc acatcctgag agtgacaaaa accctgaggg agaaggcagc tgtgccgggc 6 0 tgaggagatg acaggggtta 80 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5' detect VH1-69 FR3 <400> 31 acagaagttc cagggcagag 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3'down detect M1116(h70)<400> 32 ccccctcttg ctctctttct 20

Claims (99)

A recombinant nucleic acid molecule comprising a modified immunoglobulin (Ig) variable (V) segment encoding an anchor-modified Ig polypeptide,
The modified Ig V segment herein refers to a nucleic acid sequence encoding an Ig signal peptide and framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment, or a variant thereof. A nucleic acid sequence encoding an anchor between nucleic acid sequences encoding
wherein the anchor modified Ig polypeptide is operatively linked to:
(i) an Ig signal peptide;
(ii) an anchor; and
(iii) comprises FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment, or variants thereof;
wherein the anchor comprises a receptor binding portion of a non-immunoglobulin polypeptide of interest that binds to a cognate receptor;
Optionally, wherein the nucleic acid molecule lacks any other V segments. The recombinant nucleic acid molecule according to claim 1 , wherein the Ig signal peptide is an Ig signal peptide from a germline Ig V segment, or a variant thereof. 3. The method according to claim 1 or 2, wherein the germline Ig V segment or variant thereof is a germline Ig heavy chain variable (V _H ) segment or variant thereof, and thus
A modified Ig V segment is a combination of a nucleic acid sequence encoding an Ig signal peptide and framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _H segment or variant thereof. A modified Ig V _H segment comprising a nucleic acid sequence encoding an anchor between the encoding nucleic acid sequences;
Anchor modified Ig polypeptides into operative linkages:
(i) an Ig signal peptide;
(ii) an anchor; and
(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _H segment or variant thereof. 4. The method according to any one of claims 1 to 3, wherein the germline Ig V segment or variant thereof is a germline human (h) V _H 1-2 segment, a germline hV _H 1-3 segment, a germline hV _H 1 -8 segments, germline hV _H 1-18 segments, germline hV _H 1-24 segments, germline hV _H 1-45 segments, germline hV _H 1-46 segments, germline hV _H 1-58 segments, reproduction germline hV _H 1-69 segments, germline hV _H 2-5 segments, germline hV _H 2-26 segments, germline hV _H 2-70 segments, germline hV _H 3-7 segments, germline hV _H 3- 9 segment, germline hV _H 3-11 segment, germline hV _H 3-13 segment, germline hV _H 3-15 segment, germline hV _H 3-16 segment, germline hV _H 3-20 segment, germline hV _H segment 3-21, germline hV _H segment 3-23, germline hV _H segment 3-30, germline hV _{H segment} 3-30-3, germline hV _H segment 3-30-5, germline hV _H 3-33 segments, germline hV _H 3-35 segments, germline hV _H 3-38 segments, germline hV _H 3-43 segments, germline hV _H 3-48 segments, germline hV _H 3-49 segments , germline hV _H segment 3-53, germline hV _H segment 3-64, germline hV _H segment 3-66, germline hV _H segment 3-72, germline hV _H segment 3-73, germline hV _H segment segment 3-74, germline hV _H 4-4 segment, germline hV _H 4-28 segment, germline hV _H 4-30-1 segment, germline hV _H 4-30-2 segment, germline hV _H 4 -30-4 segments, germline hV _H 4-31 segments, germline hV _H 4-34 segments, germline hV _H 4-39 segments, germline hV _H 4-59 segments, germline hV _H 4-61 segments , a germline hV _H 5-51 segment, a germline hV _H 6-1 segment, a germline hV _H 7-4-1 segment, a germline hV _H 7-81 segment, or a variant thereof. 5. The method according to any one of claims 1 to 4, wherein the germline Ig V segment or variant thereof is a germline hV _H 1-69 segment or variant thereof, optionally wherein the Ig signal peptide is the sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7) Including, recombinant nucleic acid molecules. 6. The method according to any one of claims 3 to 5, with an operable linkage and 5' to 3':
(I) a modified Ig V _H segment;
(II) one or multiple Ig heavy chain diversity (D _H ) segments, and
(III) a recombinant nucleic acid molecule comprising one or a plurality of Ig heavy chain linking (J _H ) segments. According to claim 6,
The one or plurality of Ig D _H segments of (II) comprises one, a plurality of, or all human Ig D _H segments;
A recombinant nucleic acid molecule, wherein the one or plurality of Ig J _H segments of (III) comprises one, a plurality of, or all human Ig J _H segments. The method according to claim 6 or 7, wherein the one or plurality of Ig D _H gene segments of (II) and the one or plurality of Ig J _H gene segments of (III) form a recombined and rearranged Ig D _H /J _H sequence. to form a 5' to 3' operative linkage of a recombinant nucleic acid molecule
Modified Ig V _H gene segments and
A recombinant nucleic acid molecule comprising a rearranged Ig D _H /J _H sequence. 9. The rearranged Ig V H /D _H /J _H sequence according to claim 8, wherein the modified Ig V _H gene segment and the rearranged Ig D _{H /} J _H sequence are recombined to encode an anchor modified Ig heavy chain variable domain. form,
wherein the anchor modified Ig heavy chain variable domain is operatively linked to:
(i) an Ig signal peptide;
(ii) an anchor; and
(iii) a recombinant nucleic acid molecule comprising FR1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _H /D _H /J _H sequence. 9. The recombinant nucleic acid molecule according to any one of claims 3 to 8, wherein the modified Ig V _H segment is an unrearranged modified Ig V _H gene segment. The recombinant nucleic acid molecule according to any one of claims 6 to 10, further comprising a nucleic acid sequence encoding an Ig heavy chain constant region ( _CH ),
wherein the nucleic acid sequence encoding Ig _CH is
(I) a modified Ig V _H segment;
(II) one or multiple Ig D _H segments, and
(III) one or more Ig J _H segments
A recombinant nucleic acid molecule, operably linked downstream of. 12. The method of claim 11, wherein the nucleic acid sequence encoding Ig _CH is an Igμ gene encoding an IgM isotype, an Igδ gene encoding an IgD isotype, an Igγ gene encoding an IgG isotype, an Igα gene encoding an IgA isotype. , and/or an Igε gene encoding an IgE isoform. 13. A recombinant nucleic acid molecule according to any one of claims 3 to 12 comprising a nucleic acid sequence encoding an anchor-modified Ig heavy chain, wherein the anchor-modified Ig heavy chain is operatively linked to:
(i) an Ig signal peptide;
(ii) an anchor;
(iii) an Ig heavy chain variable domain comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _H /D _H /J _H sequence, and
(iv) a recombinant nucleic acid molecule comprising an Ig _CH . 14. The recombinant nucleic acid molecule according to any one of claims 11 to 13, wherein the Ig _CH is a non-human Ig _CH . 15. The recombinant nucleic acid molecule of claim 14, wherein the non-human Ig _CH is a rodent Ig _CH . 16. The recombinant nucleic acid molecule of claim 15, wherein the non-human Ig _CH is a rat Ig _CH . 16. The recombinant nucleic acid molecule of claim 15, wherein the non-human Ig _CH is a mouse Ig _CH . 3. The method according to claim 1 or 2, wherein the germline Ig V gene segment or variant thereof is a germline Ig light chain variable (V _L ) segment or variant thereof, and thus
The modified Ig V segment combines a nucleic acid sequence encoding an Ig signal peptide with framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _L segment or variant thereof. A modified Ig V _L segment comprising a nucleic acid sequence encoding an anchor between the encoding nucleic acid sequences;
Anchor modified Ig polypeptides into operative linkages:
(i) an Ig signal peptide;
(ii) an anchor; and
(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V _L segment or variant thereof. 19. The method of claim 18, wherein the operable linkage is 5' to 3':
(I) a modified Ig V _L segment, and
(II) a recombinant nucleic acid molecule comprising one or a plurality of Ig light chain linking (J _L ) segments. 20. The method of claim 19, wherein the modified Ig V _L segment and one or more Ig J _L segments are recombined to form a rearranged Ig V _L /J _L sequence encoding an anchor modified Ig light chain variable domain;
Anchor modified Ig light chain variable domains with an operative linkage:
(i) an Ig signal peptide;
(ii) an anchor; and
(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _L /J _L sequence. The recombinant nucleic acid molecule according to claim 19 or 20, further comprising a nucleic acid sequence encoding an Ig light chain constant region ( _CL ),
The nucleic acid sequence encoding Ig C _L is:
(I) modified Ig V _L segments and
(II) one or multiple Ig light chain linkage (J _L ) segments
A recombinant nucleic acid molecule operably linked downstream of. 22. A recombinant nucleic acid molecule according to any one of claims 18 to 21 comprising a nucleic acid sequence encoding an anchor-modified Ig light chain, wherein the anchor-modified Ig light chain is operatively linked to:
(i) an Ig signal peptide;
(ii) an anchor;
(iii) an Ig light chain variable domain comprising FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 encoded by the rearranged Ig V _L /J _L sequence, and
(iv) a recombinant nucleic acid molecule comprising an Ig C _L. 23. The recombinant nucleic acid molecule according to claim 21 or 22, wherein the Ig C _L is a non-human Ig C _L. 24. The recombinant nucleic acid molecule of claim 23, wherein the non-human Ig C _L is a rodent Ig C _L. 24. The recombinant nucleic acid molecule of claim 23, wherein the non-human Ig C _L is a rat Ig C _L. 24. The recombinant nucleic acid molecule of claim 23, wherein the non-human Ig C _L is a mouse Ig C _L. 27. The method according to any one of claims 18 to 26, wherein the germline Ig V _L segment or variant thereof is a germline Ig light chain variable kappa (Vκ) segment or variant thereof, and thus
The modified Ig V segment comprises a nucleic acid sequence encoding a signal peptide and a framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 encoding a germline Ig Vκ segment or variant thereof. A modified Ig Vκ segment comprising a nucleic acid sequence encoding an anchor between the nucleic acid sequences;
Anchor modified Ig polypeptides into operative linkages:
(i) an Ig signal peptide;
(ii) an anchor; and
(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig Vκ segment or variant thereof. 28. The method of claim 27, in an operable linkage 5' to 3':
(I) modified Ig Vκ segments, and
(II) A recombinant nucleic acid molecule comprising one or a plurality of Ig light chain binding kappa (Jκ) segments. 29. The recombinant nucleic acid molecule of claim 28, further comprising a nucleic acid sequence encoding an Ig light chain constant kappa region (CK),
wherein the nucleic acid sequence encoding the Ig CK is:
(I) modified Ig Vκ segments, and
(II) one or multiple Ig Jκ segments
A recombinant nucleic acid molecule, operably linked downstream of. 27. The method according to any one of claims 18 to 26, wherein the germline Ig V _L segment or variant thereof is a germline Ig light chain variable lambda (Vλ) segment or variant thereof, and thus
The modified Ig V segment encodes a nucleic acid sequence encoding an Ig signal peptide and framework region (FR) 1, complementarity determining region (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig Vλ segment or variant thereof. A modified Ig Vλ segment comprising a nucleic acid sequence encoding an anchor between nucleic acid sequences that
Anchor modified Ig polypeptides into operative linkages:
(i) an Ig signal peptide;
(ii) an anchor; and
(iii) a recombinant nucleic acid molecule comprising FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig Vλ segment or variant thereof. 31. The method of claim 30, 5' to 3' in an operable linkage:
(I) Modified Ig Vλ segments, and
(II) A recombinant nucleic acid molecule comprising one or a plurality of Ig light chain binding lambda (Jλ) segments. 32. The recombinant nucleic acid molecule according to claim 31, further comprising a nucleic acid sequence encoding an Ig light chain constant lambda region (Cλ),
The nucleic acid sequence encoding Ig Cλ is:
(I) Modified Ig Vλ segments, and
(II) one or more Ig Jλ segments
A recombinant nucleic acid molecule, operably linked downstream of. 33. The method of any one of claims 1-32, wherein the anchor binds the receptor binding portion of the non-immunoglobulin polypeptide of interest to the FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment, or variants thereof. A recombinant nucleic acid molecule comprising a linker that connects to. 34. The recombinant nucleic acid molecule of claim 33, wherein the linker comprises the sequence GGGGS (SEQ ID NO: 5). 35. The recombinant nucleic acid molecule according to any one of claims 1 to 34, wherein the anchor comprises a natriuretic peptide receptor (NPR) binding portion of a natriuretic peptide (NP). 36. The recombinant nucleic acid molecule of claim 35, wherein the NPR binding portion of NP comprises the C-terminal tail of NP. 37. The recombinant nucleic acid molecule of claim 35 or 36, wherein the NP is atrial natriuretic peptide (ANP). 38. The recombinant nucleic acid molecule according to any one of claims 1 to 37, wherein the anchor comprises the sequence NSFRY (SEQ ID NO: 3). 39. The sequence according to any one of claims 1 to 38, presented as SEQ ID NO: 8 or a degenerate variant thereof, the sequence presented as SEQ ID NO: 10 or a degenerate variant thereof, SEQ ID NO: 11, and SEQ ID NO: 12 or a degenerate variant thereof A recombinant nucleic acid molecule comprising a sequence selected from the group consisting of degenerate variants. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 10 and 33 to 39, wherein the targeting vector, upon homologous recombination between the targeting vector and a non-human Ig heavy chain locus, targets 5' and 3' homology targeting the non-human Ig heavy chain locus to include a recombinant nucleic acid molecule in an operative linkage upstream of the non-human Ig _CH at the non-human Ig heavy chain locus. further comprising cancer, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus, and/or the non-human Ig heavy chain locus is a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V _H , D _H , and/or deletion of the _JH gene segment, or a combination thereof. 41. The targeting vector of claim 40, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule substitutes the non-human V _H segment at the non-human Ig heavy chain locus. 42. The method of claim 40 or 41, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule comprises one or more non-human V H segments, all non-human V _H segments at the non-human Ig heavy chain locus. A targeting vector that substitutes the D _H segment, and all non-human J _H segments. 43. The method according to any one of claims 40 to 42, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule comprises one non-human V _H segment at the non-human Ig heavy chain locus or A targeting vector that substitutes all but all non-human V _H segments, all non-human D _H segments, and all non-human J _H segments. 44. The method according to any one of claims 40 to 43, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus is transferred from the non-human Ig heavy chain locus to the non-human Ig heavy chain locus. A targeting vector comprising a recombinant nucleic acid molecule in operative linkage to regulatory sequences. 45. The targeting vector of any one of claims 40-44, wherein the 5' homology arm comprises the sequence presented as SEQ ID NO: 11 and/or the 3' homology arm comprises the sequence presented as SEQ ID NO: 12. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 17 and 33 to 39, wherein the targeting vector upon homologous recombination between the targeting vector and a non-human Ig heavy chain locus, 5' and 3' homology targeting the non-human Ig heavy chain locus such that the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule in operable linkage to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus. further comprising cancer, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or the non-human Ig heavy chain locus is a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V _H , D _H , and/or deletion of the _JH gene segment, or a combination thereof. 47. The method of claim 46, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule comprises at least one non-human V _H segment, all non-human D _H gene segments at the non-human Ig heavy chain locus , a targeting vector that replaces all non-human J _H gene segments, and one or more non-human C _H genes. Claims 1 to 2, 18 to 20, 27 to 28, 30 to 31, and claims 33 to 38 comprising the recombinant nucleic acid molecule of any one of A targeting vector, wherein, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus operates upstream of the non-human Ig C _L at the non-human Ig light chain locus. optionally further comprising 5' and 3' homology arms targeting the non-human Ig light chain locus to include the recombinant nucleic acid molecule in possible linkage, wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or The targeting vector of claim 1 , wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig V _L and/or J _L gene segments, or a combination thereof. 49. The targeting vector of claim 48, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule substitutes the non-human V _L segment at the non-human Ig light chain locus. 50. The method of claim 48 or 49, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule comprises at least one non-human V L segment and all non-human V _L segments at the non-human Ig light chain locus. A targeting vector, substituting the J _L segment. 51. The method of any one of claims 48 to 50, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule comprises all non-human V _L segments and all non-human Ig light chain loci at the non-human Ig light chain locus. A targeting vector, substituting the non-human J _H segment. 52. The method according to any one of claims 48 to 51, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus is a non-human Ig light chain regulatory sequence at the Ig light chain locus. A targeting vector comprising a recombinant nucleic acid molecule in operative linkage to a targeting vector. 39. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1-2, 18-38, wherein the targeting vector upon homologous recombination between the targeting vector and a non-human Ig light chain locus, the targeting vector 5' and 3' homology arms targeting the non-human Ig light chain locus such that the modified non-human Ig light chain locus contains a recombinant nucleic acid molecule in operable linkage to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus. optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or the non-human Ig light chain locus is a human or humanized immunoglobulin light chain variable region, endogenous Ig V _L and/or J _L A targeting vector comprising a deletion of a gene segment, or a combination thereof. 54. The method of claim 53, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule comprises a non-human V L segment, all non-human J _L gene segments, and all non-human J _L gene segments at the non-human Ig light chain locus. A targeting vector that substitutes for the non-human C _L gene. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 20, 27 to 28, and 33 to 38, wherein the targeting vector is Upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus is recombined with an operable linkage upstream of the non-human Ig CK at the non-human Ig light chain κ locus. optionally further comprising 5' and 3' homology arms targeting the non-human Ig light chain κ locus to include, wherein the non-human Ig light chain κ locus is an endogenous rodent Ig light chain κ locus and/or is a non-human The targeting vector of claim 1 , wherein the Ig light chain κ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig Vκ and/or Jκ gene segments, or a combination thereof. 56. The targeting vector of claim 55, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule substitutes a non-human VK segment at the non-human Ig light chain κ locus. 57. The method of claim 55 or 56, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule comprises at least one non-human Vκ segment and all non-human Vκ segments at the non-human Ig light chain κ locus. A targeting vector substituting the human Jκ segment. 58. The method of any one of claims 55-57, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule is directed to all non-human Vκ segments at the non-human Ig light chain κ locus. and a targeting vector that substitutes for all non-human Jκ segments. 59. The method of any one of claims 55-58, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the targeted non-human Ig light chain κ locus is selected from the Ig light chain κ locus to the non-human Ig A targeting vector comprising a recombinant nucleic acid molecule in operative linkage to a light chain κ regulatory sequence. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2 and 18 to 29, wherein the targeting vector undergoes homologous recombination between the targeting vector and a non-human Ig light chain κ locus. , 5' and 3' targeting the non-human Ig light chain κ locus such that the targeted non-human Ig light chain κ locus comprises a recombinant nucleic acid molecule in an operable linkage to a non-human Ig light chain κ regulatory sequence at the Ig light chain κ locus. A targeting vector, further comprising a homology arm. 61. The method of claim 60, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain κ locus, the recombinant nucleic acid molecule comprises a non-human VK segment at the non-human Ig light chain κ locus, all non-human JK gene segments, and A targeting vector that substitutes for a non-human CK gene. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 20, 30 to 31, and 33 to 38, wherein the targeting vector Upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus converts the recombinant nucleic acid molecule into an operable linkage upstream of the non-human Ig Cλ at the non-human Ig light chain locus. optionally further comprising 5' and 3' homology arms targeting the non-human Ig light chain λ locus to include, wherein the non-human Ig light chain λ locus is an endogenous rodent Ig light chain λ locus and/or is a non-human Ig The targeting vector of claim 1 , wherein the light chain λ locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of an endogenous Ig Vλ and/or Jλ gene segment, or a combination thereof. 63. The targeting vector of claim 62, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule substitutes a non-human Vλ segment at the non-human Ig light chain λ locus. 64. The method of claim 62 or 63, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule comprises at least one non-human Vλ segment and all non-human Vλ segments at the non-human Ig light chain locus. A targeting vector, substituting the Jλ segment. 65. The method of any one of claims 62 to 64, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule comprises all non-human Vλ segments at the non-human Ig light chain λ locus and A targeting vector, substituting all non-human Jλ segments. 66. The method of any one of claims 62 to 65, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the targeted non-human Ig light chain λ locus is a non-human Ig at the Ig light chain λ locus. A targeting vector comprising a recombinant nucleic acid molecule in operative linkage to a light chain λ regulatory sequence. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 26, and 30 to 38, wherein the targeting vector comprises a targeting vector and a non-human Ig light chain. Upon homologous recombination between the λ loci, the non-human Ig light chain λ locus of the targeted non-human Ig light chain λ locus comprises a recombinant nucleic acid molecule in an operable linkage to the non-human Ig light chain λ regulatory sequence at the Ig light chain λ locus. A targeting vector, further comprising 5' and 3' homology arms targeting. 68. The method of claim 67, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain λ locus, the recombinant nucleic acid molecule comprises a non-human Vλ segment at the non-human Ig light chain λ locus, all non-human Jλ gene segments, and A targeting vector that substitutes for a non-human Cλ gene. A non-human animal genome comprising the recombinant nucleic acid molecule of any one of claims 1-39 or the targeting vector of any one of claims 40-68, optionally wherein the non-human animal is a rodent, wherein the rodent is a rat or mouse, a non-human animal genome. 70. The non-human animal genome of claim 69, wherein the recombinant nucleic acid is at an endogenous Ig locus of the non-human animal genome. A non-human animal comprising the recombinant nucleic acid molecule of any one of claims 1 to 39, the targeting vector of any one of claims 40 to 68, or the non-human animal genome of claims 69 or 70, or non-human animal cells. 72. The non-human animal or non-human animal cell of claim 71, wherein the recombinant nucleic acid molecule, targeting vector, or non-human animal genome is in the germline of the non-human animal or non-human animal cell. 40. An in vitro method of modifying an isolated cell comprising introducing a recombinant nucleic acid molecule of any one of claims 1-39 into the isolated cell. 74. The in vitro method of claim 73, wherein introducing comprises contacting the cell with the targeting vector of any one of claims 40-68. 75. The in vitro method of claim 73 or 74, wherein the cell is a host cell. 75. The in vitro method of claim 73 or 74, wherein the cell is an embryonic stem (ES) cell. 77. The in vitro method of any one of claims 73-76, wherein the cells are rodent cells, optionally the rodent cells are rat cells or mouse cells. A non-human animal embryo generated from the embryonic stem cell of claim 76 . A non-human animal generated from the embryonic stem cell of claim 76 . Preparation of a non-human animal comprising implanting the ES cell or embryo comprising the ES cell of claim 76 into a suitable host and maintaining the host under suitable conditions while the ES cell or embryo develops into a viable progeny. method. The non-human animal of any one of claims 71-72 and 79 or the non-human animal prepared according to the method of claim 80, wherein the non-human animal compared to a control non-human animal:
(a) comparable numbers of mature B cells in the spleen;
(b) comparable numbers of kappa-positive B cells in the spleen;
(c) comparable numbers of lambda positive B cells in the spleen;
(d) comparable levels of serum IgG and/or
(e) non-human animals with comparable levels of serum IgM. The non-human animal of any one of claims 71-72, 79 or 81, or a non-human animal prepared according to the method of claim 80, wherein the non-human animal is a control non-human animal A non-human animal capable of receiving an immune-response comparable to that of In the non-human animal of any one of claims 71 to 72 and 79 and 81 to 82 or a non-human animal prepared according to the method of claim 80, each anchor-modified Ig polypeptide and optionally a plurality of antigen-binding proteins comprising:
wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or
wherein the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry;
wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, and the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry, non-human animal. The non-human animal of any one of claims 71-72 and 79 and 81-83 or the non-human animal prepared according to the method of claim 80, wherein the non-human animal is of interest A non-human animal further comprising a cognate receptor for a non-immunoglobulin polypeptide. In the non-human animal of any one of claims 71 to 72 and 79 and 81 to 84 or the non-human animal prepared according to the method of claim 80, each anchor-modified Ig polypeptide and a plurality of antigen-binding proteins that specifically bind to a cognate receptor of a non-immunoglobulin polypeptide of interest, optionally:
wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or
wherein the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry;
wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide and the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry. . 86. The non-human animal of claim 84 or 85, wherein the cognate receptor is a natriuretic peptide receptor (NPR). 87. The non-human animal of any one of claims 84-86, wherein each of the plurality of antigen-binding proteins comprises a KD of less than 1X10 ⁹ and/or a t½ of greater than 30 minutes. 88. The non-human animal of any one of claims 84-87, wherein at least 15% of the plurality of antigen-binding proteins block binding of a cognate receptor to a non-immunoglobulin polypeptide of interest. 89. The non-human animal of any one of claims 84-88, wherein greater than 50% of the plurality of antigen-binding proteins bind to cognate receptors expressed on the cell surface. The non-human animal of any one of claims 71-72, 79, and 81-89, or a non-human animal prepared according to the method of claim 80, wherein the non-human animal A non-human animal that is a rodent, optionally wherein the rodent is a rat or mouse. A method of producing an antigen-binding protein or obtaining a nucleic acid encoding it, the method comprising:
immunizing the non-human animal of any one of claims 71 to 72, 79, and 81 to 90, or a non-human animal prepared according to the method of claim 80, with an antigen;
enabling a non-human animal to produce an antigen-binding protein comprising an anchor-modified Ig polypeptide that binds an antigen or a nucleic acid encoding the same, and optionally:
wherein the mass of the antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or
wherein the mass of the antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry;
wherein the mass of the antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, and wherein the mass of the antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry. 92. The method of claim 91, further comprising recovering the antigen binding protein or nucleic acid encoding it from the non-human animal or non-human animal cell. 93. The method of claim 92, wherein the non-human animal cell is a B cell or hybridoma. A non-human animal cell recovered according to the method of claim 91 . 95. The non-human animal of claim 94, wherein the non-human animal cell is a B cell. 96. The non-human animal cell of claim 94 or 95, wherein the B cell is a mouse B cell. A hybridoma cell comprising the non-human animal cell of any one of claims 94-95 fused with a myeloma cell. encoded by the recombinant nucleic acid molecule of any one of claims 1 to 39, the targeting vector of any one of claims 40 to 68, and the non-human animal genome of any one of claims 69 to 70; The method of any one of claims 73 - 76 - 80 expressed by the non-human animal or non-human animal cell of any one of claims 71 - 72 and 81 - 90 . An anchor-modified Ig polypeptide expressed by a non-human animal or non-human animal cell prepared according to, or prepared according to the method of any one of claims 91-93, optionally:
wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, or
wherein the mass of each antigen-binding protein is determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry;
wherein the mass of each antigen-binding protein confirms the presence of an anchor-modified Ig polypeptide, wherein the mass of each antigen-binding protein is anchor-modified, determined by matrix-assisted laser desorption ionization - time-of-flight mass spectrometry. Ig polypeptide. 99. The anchor-modified Ig polypeptide of claim 98 comprising at its N-terminus the amino acid sequence set forth in SEQ ID NO:3.